The fatwas issued on organ transplantation primarily concern the permissibility donations from living and deceased donations. When organ transplantation first became medically possible, public debates on the topic in the Muslim world were controversial. Organ transplantation was thought to undermine the traditional legal understanding of life and death. Today, the majority opinion of Muslim legal scholars is in favour of organ transplantation. Legal scholars agree that living and deceased human beings have an inherent *ḥurma* (dignity) which protects their bodies from interference. However, this inviolability can be overridden, and organ transplantation is justified if other legal-ethical principles are given preference.

The principles that justify deceased and living organ transplantation are *ḍarūra* (necessity), which renders a prohibited act permissible in case of severe duress, and *farḍ kifāya,* which imposes the duty to save a life on every Muslim. Muslim scholars eventually accepted that the concept of brain death is in accordance with Islamic law. Muslim jurists mainly argue that death has occurred when the body can no longer respond and serve the soul. Brain death is a form of this state. Furthermore, Muslim jurists argue that the criteria and diagnosis of death depend on human experience because Islamic sources have not defined death. Therefore, it is appropriate from a religious perspective to apply the death criteria accepted in the medical sciences, which includes brain death as a form of death.

Organ transplantation has also been treated from ethical and legal points of view in academic contexts and by international legal bodies and various bioethical councils to determine how organ transplantation should be regulated. The leading principles of bioethics are the principles of autonomy, beneficence, nonmaleficence, and equity. These principles have informed various legal documents on bioethics, such as the UNESCO Universal Declaration on Bioethics and Human Rights (UDBHR). The *WHO Guiding Principles on Human Organ Transplantation* is the legal-ethical document most internationally relevant to organ transplantation. The *WHOGuiding Principles* were first endorsed in 1991 to provide an ethical framework for the acquisition and transplantation of human organs. The *Guiding Principles* emphasize preserving the donor's life and health, voluntary and informed consent, noncommercial donation, and preferences for deceased over living donation and related living over unrelated living donation.

This analysis of the laws of Middle East and North Africa (MENA) states has shown that the states have kept pace with developments in organ transplantation. Concurrently with the development of organ transplantation as a therapeutic method, the states in the MENA region started to legislate on the issue. The pieces of legislation were influenced by the ethical-legal discussions in Islamic jurisprudence, in academia, and in various bioethical councils and international organizations. Therefore, these pieces of legislation are an amalgam of Islamic ethical-legal elements and internationally formed ethical-legal standards. The elements in the legislation that can be seen as rooted in fatwas are the permission of living and deceased donation to save another person's life, which is an expression derived from the Islamic legal principle of *farḍkifāya* and the legal principle of *ḍarūra;* the prohibition of living organ donation if it endangers the life of the donor, derived from the Islamic legal prohibition of self-harm and suicide; the acceptance of brain death criteria, arising from the opinion that Islamic legal tradition leaves the definition of death to medical experts due to the absence of a definition in the Islamic source; and the prohibition of donation of reproductive organs because of the danger of 'mixing genealogy', which contradicts the *maqāṣid al-sharīʿa* to preserve Islamic family values, including the preservation of legitimate lineage according to Islamic family law.

Elements that are especially prominent in the *WHOGuiding Principles on Human Organ Transplantation* that are also found in the legislation examined are free and informed consent for living donation, the prohibition of the sale and purchase of organs, and the preference for living related donation over unrelated living donation.

Although certain elements of regulation are more important to Islamic law and others are more important to the *WHOGuiding Principles,* many elements are essential to both systems. For example, the equivalent to the requirement of *ḍarūra* for organ transplantation to save another person's life in Islamic law is the requirement expressed in the *WHOGuiding Principles* that organ transplantation needs to have a therapeutic effect and must not harm the donor's life or health. Likewise, the requirement of noncommercial donation emphasized in the *WHOGuiding Principles* is also found in the fatwas. Some regulations seem to have been informed by both Islamic law and common international bioethical standards, such as the regulations on brain-death donation: Islamic law permits organ transplantation from brain-dead donors by accepting brain death as a form of death.

Lastly, some elements that are discussed in fatwas or required by the *WHO Guiding Principles* are not well received in the regulations of the examined states. At the beginning, most Islamic legal scholars opposed deceased donation due to the concept of bodily resurrection after death in Islamic legal tradition. Organ removal was thought to break the requirements of Islamic burial rites, in which a corpse needs to be buried intact because the body is needed for resurrection after death. Although this issue was the subject of long and controversial debates, only a few states explicitly legislated that the corpse needs to be rendered to its original state externally after the removal of the organ. One might have expected that more states would have legislated similar requirements to fulfil some Islamic legal opinions. Another issue that is not widely addressed in the legislation is the equitable allocation of organs stipulated in the *WHOGuiding Principles.* Although not discussed widely in prominent fatwas on organ transplantation, just and fair allocation is also a requirement for transplantation from an Islamic point of view. It can only be assumed that MENA legislature have not addressed this issue in their laws but in other documents, such as guidelines for medical professionals' practice.

#### **II. Answers to Research Question**

The literature on Islamic law finds that the laws today in states with an Islamic legal background were mostly replaced by European or European-inspired laws during colonization in the 18th and 19th centuries, except for personal status law. The states later codified personal status laws in line with classical Islamic law, which had been developed and applied by jurists during the centuries before European colonization. The literature also finds that the fields of law that have emerged in response to technological and scientific advances, such as organ transplantation in the medical field, follow an internationally set legal standard and, thus, are not based on religious law.

The examination of organ transplantation laws in Muslim-majority states of the MENA region has shown that organ transplantation laws are an amalgam of Islamic legal norms and the international standard rules derived from the common bioethical principles represented in the *WHOGuiding Principles on Human Organ Transplantation.* The analysis presented here indicates that organ transplantation laws in MENA states do not fall into the category of laws based on classical Islamic law, nor do they fall entirely into the category of laws with no connection to Islamic law. The research has shown that laws regulating novel issues are shaped by Islamic and non-Islamic legal elements.

The reason for using the specific wordings derived from fatwas might be a desire to show that Islamic law was incorporated in the regulations and that the regulations accord with Islamic law. This would support the view that sharia continues to play a programmatic rather than substantial role in legislation processes, including in laws on novel subjects. Sharia is used in this way to give laws a higher legitimization. The legislator also uses sharia to give laws a certain quality that Muslim societies demand: that the laws represent their identity through the acknowledgement of their legal culture, even though today's laws are no longer derived from classical Islamic laws but are legislated by the state (and not God), the only legitimate lawgiver in modern states.

The analysis presented here indicates that the influence of Islamic law seems to be restricted to the permissibility of organ transplantation while neglecting other issues that are discussed in fatwas, such as the question of the state of the corpse after organ removal, which is a critical aspect in Islamic law on rituals. Furthermore, the Islamic legal-ethical elements incorporated in the regulations are congruent with general bioethics as represented in the *WHO Guiding Principles.* It can be argued that the regulations on organ transplantation would have remained largely similar even without reference to the elements extracted from fatwas by following the wording of the *WHO Guiding Principles.* Two examples are that organ transplantation must follow therapeutic purposes and that the health of the donor must be protected throughout the process, which are expressed in the legislation of many MENA states as the requirement of necessity to save another person's life and the prohibition of removing a vital organ.

In any case, the prevalent characterization of Islamic law found in the literature cannot be confirmed for organ transplantation laws. Organ transplantation laws have no grounding in classical Islamic law, but they are not entirely detached from Islamic law. It is conceivable that this finding can be applied to other fields of legislation following the same logic. Because Islamic law claims to regulate all aspects of life and lay Muslims are interested in knowing and following Islamic legal rulings on the various aspects of their lives so that they can live by Islamic law, fatwas are given on every topic. Legislators then acknowledge the opinions published in fatwas by producing regulations that are at least congruent with Islamic legal rulings. This finding indicates that the belief popularized by Islamist movements, that the majority of laws in MENA states do not conform to sharia, is erroneous, at least for the issue of organ transplantation. However, further research analysing novel laws is needed to make similar statements about other fields of law.

#### **III. Further Results**

This analysis of organ transplantation laws in Muslim-majority states of the MENA region does not support the view that a 'closing of the door of *ijtihād*' has occurred in Sunni Islam.**590** This work has shown that Muslim legal scholars have exercised *ijtihād* on the topic of organ transplantation, a subject that the primary Islamic legal sources did not cover and on which no precedent ruling exists in the Islamic legal tradition and have nonetheless developed rulings derived from Islamic law. Furthermore, the argument that Islamic law no longer exists since the rise of the modern nation-state system cannot be confirmed either, because numerous fatwas have been published on the issue of organ transplantation. These fatwas are authoritative and have a binding effect on the Muslim believer even though they are not enforced by the state and, thus, are not legally binding.

A further conclusion concerns the phenomenon labelled re-Islamization in the literature. The literature finds that it is mainly criminal law that has been affected by the re-Islamization process because some states have re-introduced some rules from classical Islamic law in their state legislations. Re-Islamization efforts are also observed in the provisions found in some states' constitutions that require the state's laws to be compatible with the sharia. These shariacompatibility provisions in constitutions have been found to have more of a 'cosmetic' nature than having had any real effect on the actual introduction of Islamic law in state legislation.**591** This examination of organ transplantation laws has shown that the popular demand that state laws have a connection to Islamic legal culture has succeeded to some extent.

# **Summary**

This research has shown that all Muslim-majority states except Iran have legislated the issue of organ transplantation for living transplantation and all except Yemen for deceased organ transplantation. All states allow living and deceased donation provided that the transplantation saves another person's life and the procedure does not endanger the donor's life. Brain death is accepted in all states as a form of death. The donor's consent is also required in all states for both living and deceased transplantation. All the regulations examined

590 Schacht, Introduction to Islamic Law, 70–71.

591 Peters, "From Jurists' Law to Statute Law," 91; Lombardi, State Law as Islamic Law, 129, 135; Lombardi, "Constitutional Provisions," 770.

require the next of kin to consent if the deceased did not express their will to donate during their lifetime. However, most laws have not explicitly addressed whether the family needs to act according to the presumed will of the deceased, although it can be assumed to be so. All states prohibit the sale and purchase of organs in their laws except for Iran. In Iran, a programme is in place for commercial living organ transplantation; the state gives the donor a monetary 'gift of altruism' and a price is negotiated privately between the donor and the recipient.

The laws in Muslim-majority states of the MENA region generally follow an internationally set standard for ethical regulation of organ transplantation. This examination of the laws has also shown that they clearly incorporate rulings from Islamic law. These rulings or legal opinions are called fatwas and are developed by Muslim legal scholars with the sources and methods of Islamic jurisprudence.  However, the influence of Islamic law is mainly restricted to the question of the permissibility of organ transplantation and, thus, whether the regulations follow sharia.

This leads to the finding that novel laws, such as organ transplantation laws, are not completely detached from the Islamic legal tradition, as claimed in the literature on Islamic law. It seems as if the idea that laws in MENA states should comply with the sharia has affected new legislation. Organ transplantation laws comply with the sharia by having incorporated rules established in fatwas and highlight this fact by employing the terminology used in these fatwas.

## *About the Author*

Arezoo Sang Bastian studied law at the University of Zurich and the University of Strasbourg. She also studied Middle Eastern Studies at the University of Zurich. She is a former research assistant at the Chair of Prof. Dr. Andrea Büchler and former project manager of the Center for Islamic and Middle Eastern Legal Studies (CIMELS) at the University of Zurich. She is currently working at a Court in Zurich.

The Faculty of Law of the University of Zurich accepted this thesis as a doctoral dissertation on the 4th of October 2023 at the request of Prof. Dr. Thomas Gächter and Prof. Dr. Brigitte Tag and awarded it the distinction *magna cum laude.*

*sui generis* is an association dedicated to the promotion of free access to legal literature, court decisions, decisions of authorities and legislative materials. Since 2014, a legal open access journal has been published under the label *sui generis.* In 2019, the *sui generis* publishing house was founded.

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Published in the *sui generis* series, edited by Marc Thommen.

1**st** edition, 31 January 2024 © 2024 Arezoo Sang Bastian *Print of the dissertation approved by the Faculty of Law of the University of Zurich.*

This work has been published under a Creative Commons license which requires only the attribution of the author when being reused (CC BY 4.0 — https://creativecommons.org/licenses/by/4.0).

Published with the support of the Swiss National Science Foundation.

ISBN: 978-3-907297-52-0 DOI: 10.38107/052 Proofreading: Dr. Simon Milligan Design: Müller+Hess, Basel Print: Ebner & Spiegel, Ulm

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# **052**

# BETWEEN ISLAMIC AND SECULAR LAW

**sg**

When describing the legal systems and laws of today's Muslim-majority states in the Middle Eastern and North African (MENA) region, two categories of laws are identified: one derived from Islamic and one from non-Islamic law. Following this notion, the literature finds that novel legal areas do not have any connection to Islamic law since they were not regulated by classical Islamic law. In contrast, the topic of organ transplantation is inherently connected to Islamic law because Islam considers itself to encompass all aspects of life. This research based on the analysis of organ transplantation laws of the MENA states encourages to rethink that a strict dichotomy between Islamic and non-Islamic laws does not exist. Organ transplantation laws in MENA states generally follow an international legal standard while also complying with the sharia.

sui generis ISBN 978-3-907297-52-0 DOI 10.38107/052


*2.3. Mechanisms through Which SMS May Influence Safety Outcomes*

SMS may lead to positive safety outcomes through at least two mechanisms. The first is that SMS provides a systematic way of managing organizational safety, and that it clearly places responsibility for this process in the company. The philosophy that the ISM code is based on is total quality management, highlighting continuous improvement through management commitment and personnel empowerment (Lappalainen 2008 in [29]). The process of continuous improvement follows Deming's circle ('plan–do–check–act') [29]. Implementing an SMS involves analyzing the safety status and setting goals (plan), implementing measures to fulfil the goals (do), developing indicators to measure goal attainment (check), and adjusting the measures if necessary (act).

The second mechanism through which SMS may produce positive safety outcomes is through the development of a positive safety culture. As noted, this is the explicit motivation for most SMS legislation, hypothesizing that the development of formal procedures ("how things should be done"), will influence informal practices ("how things actually are done") [11]. Implementing an SMS largely concerns implementing certain routines and ways of doing things in the organization, e.g., carrying out risk assessments, documenting the process, developing procedures, and training new employees etc. [13,16,17]. The implementation of such activities can lead to improved safety culture, especially if it is followed by increased focus on safety among managers and employees in the organization.

#### *2.4. Potential Barriers to SMS Implementaion*

Contrary to the hypothesized relationship between SMS and safety culture that is described above, several studies report discrepancies between formal and informal aspects of safety in organizations [26,33,34]. Thus, it seems that a positive safety culture does not necessarily follow from SMS implementation. This can be explained by, e.g., SMS that are poorly adapted to the companies in question, by referring to the nature of the working activities, to the competence of the personnel, etc. [26,34]. These factors can also be related to the size and maturity of companies, etc. The report from the ITF [22] roundtable on SMS in transport mentions several such potential barriers to successful SMS implementation in the road sector, for example: (1) there is no international regulatory or standard setting body in road transport of the kind responsible for aviation (ICAO) or maritime transport (IMO); (2) there is a large share of non-professional road users; (3) there are many small companies; (4) high proportion of owner-drivers; and (5) low organizational maturity, indicated, e.g., by low degree of reporting of incidents and little systematic focus and follow up of indicators measuring performance [22]. These factors may challenge the possibilities to implement SMS and continuous improvement (PDCA) in companies in the road sector.

#### *2.5. Hypotheses*

As the SMS rules in the maritime sector firmly places the responsibility for safety on the shipping company and the shipmaster, we hypothesize that respondents in the maritime sector have a stronger perception that the responsibility for accident prevention is clearly defined in their sector regulations (Hypothesis 1). Moreover, based on this we also expect respondents in the maritime sector to rate their efforts to prevent work related accidents as higher than respondents in the road sector (Hypothesis 2). Finally, we expect that respondents in the maritime sector rate SMS as more important for the safety level in their sector than respondents in the road sector (Hypothesis 3).

#### **3. Methods**

We have used two methods to fulfil the study aims. The first method is semi structured qualitative interviews with 17 sector experts. We conducted these to get rich information, based on the interviewees' experiences and viewpoints, and also as a background to the development of the quantitative survey. The qualitative interviews resulted in textual data that we used to complement the numerical data from the survey. The use of quantitative survey data is the second method, and this is based on 112 respondents. We use this to make statistical comparisons between the answers of respondents from the two sectors.

#### *3.1. Interviews*

We have conducted qualitative interviews with 17 sector experts to gain knowledge on the aims of the study. Interviewees were selected from transport companies, the Labour Inspection Authority, the Public Roads Administration, including personnel from the Accident Analysis Groups, the Norwegian Maritime Directorate, the Accident Investigation Board, the Norwegian Coastal Administration, and other relevant actors. The interviews were conducted in 2016 and 2017. The interviews were conducted face-to-face and by telephone. The interviews generally lasted for between one and one and a half hours. We used a semi structured interview guide, and the themes and questions in the guide focused on the aims of the study.

The interview guide focused on the following themes:

The purpose of the interviews was to give us more insight into the interviewees' understanding of their organizations' knowledge and information sources about work-related accidents, views on risk factors and thoughts on roles and responsibilities. We collected this information to be able to conduct rich comparisons between the studied sectors. Interviewees were encouraged to "think out loud" and they were assured that the purpose of the interview was to provide us with nuanced viewpoints and thoughts that we cannot collect by means of survey methods. Although the themes in the interviews were fairly similar to those in the survey, the qualitative interviews involved open ended questions which allowed the interviewees to elaborate freely when answering.

#### *3.2. Survey*

#### 3.2.1. Recruitment of Respondents and Sample Characteristics

A small-scale web-based survey was distributed to representatives from government agencies, NGOs, and personnel in transport companies in the two sectors. Relevant representatives from government agencies were identified with assistance from the project's reference group. The survey was sent to relevant persons in the Transport Accident Inves-

tigation Board Norway, the Ministry of Transport and Communications, the Norwegian Public Road Authority, the Norwegian Maritime Authority, the Norwegian Labour Inspection Authority, and the Norwegian Public Roads Administration's Accident Analysis Groups. The survey was also sent to transport companies in the studied sectors, and they were asked to distribute the survey among elected safety officials and trade union representatives. Thus, the survey includes what we may term sector experts, with good knowledge resulting from efforts to prevent work-related accidents within the sectors. Table 1 shows the distribution of sector experts in the different sectors, by type of organization.

**Table 1.** Distribution of sector experts in the studied sectors, by type of organization.

There were 112 individual respondents: 59% (N = 66) from the road sector and 41% (N = 46) from the maritime sector. The respondents from transport companies in both sectors were largely recruited from two unions: one union from the road sector including drivers and one union from the maritime sector which largely includes ship navigators. Thus, although both groups of union members are transport operators, the operators from the maritime sector rank higher in the organizational hierarchy than those in the road sector. While the method of distribution makes it impossible to calculate the response rate, it is obvious that it has been very low. This, in addition to the limited number of responses, means that results must be treated with considerable caution.

#### 3.2.2. Survey Themes

The survey contains seven different themes:

#### 3.2.3. Quantitative Analysis

Regression analysis. We have performed two regression analyzes. The first was conducted to assess the conditions explaining variation in the respondents' answer to the question: "Is responsibility for accident prevention sufficiently clearly defined in current regulations in the sector?" The second was to assess respondents' agreement with the statement: "How would you rate your organization's efforts to prevent work-related accidents?". We used logistic regression in the first analysis, as the dependent variable was made dichotomous (1: Yes, 2: No/do not know). We used linear regression in the second analysis, since the dependent variables are continuous. The regression analyzes show the effects of the independent variables that we include on the two dependent variables, controlled for the other variables in the analysis.

#### **4. Results**

#### *4.1. Is Responsibility for Accident Prevention Is Clearly Defined?*

The first aim of the study was to examine how the different sector rules influence perceptions of whether the responsibility to prevent work-related accidents is clearly defined in the maritime sector and in the road sector. As the SMS rules in the maritime sector firmly places the responsibility for safety on the shipping company and the shipmaster, we hypothesize that respondents in the maritime sector have a stronger perception that the responsibility for accident prevention is clearly defined in their sector regulations (Hypothesis 1).

In the survey, we asked whether responsibility for accident prevention was defined clearly enough in current regulations in the sector. The road sector stands out with a relatively low share of respondents stating that responsibility is defined clearly enough; 37% answered yes, compared with 78% in the maritime sector. The corresponding shares for no were 31% in road and 13% in the maritime sector. The road sector also had a larger percentage of respondents stating that they "do not know" (31%), compared with 9% in the maritime sector. Differences between the sectors were statistically significant at the 1% level.

Respondents from the road sector who gave reasons for their answers referred to the relationship between drivers and their employer organizations and/or their customers. They referred to stress or pressure as a result of short deadlines, and the fact that drivers are held responsible even though they are not the ones who define the route and the time frame. In this manner, they underlined that transport buyers also indirectly influence the safety of transport assignments. Moreover, they also pointed to drivers' legal responsibilities as a problem, as they may provide an opportunity for managers or customers to ignore their actual influence and liability. This indicates a discrepancy between the legal responsibilities of drivers and the practical responsibilities of, e.g., transport buyers in the road sector.

The majority of interviewees from the road sector thought employers should take more responsibility for their employees' behaviour and safety in traffic, as part of their HSE work. Specifically, they believed that work assignments should be based on risk assessments, including an analysis of the road and conditions, and employers should develop safetypromoting routines, procedures and guidelines. It was noted that many businesses fail to fulfil these requirements today. Road transport differs from most professional sectors in that individual drivers usually carry the entire responsibility for safety, and some of the interviewees stated that the companies have—or should take on— the overarching responsibility for the interplay between the various elements and actors involved in road traffic. Some of the interviewees from the road sector believed that relations of responsibility in this area should be more clearly defined, especially HSE-requirements or safety standards. Others found responsibilities to be well-defined in theory, but they said that the practical follow-up was inconsistent. The Working Environment Act gives employers a wide-ranging responsibility for their workers' safety, but interviewees noted that this is rarely enforced. The Road Traffic Act—which places all responsibility with the driver—is enforced through controls and in police investigations following accidents, but the responsibility of the firms is not enforced to the same degree. So while the law divides responsibility between different actors, this is not the case in practice.

Interviewees in the maritime sector underline that safety in the maritime sector is circumscribed by the International Maritime Organisation (IMO). This is a specialized agency of the United Nations whose primary purpose is to develop and maintain a comprehensive regulatory framework for shipping. The Norwegian Maritime Authority (NMA) is the government agency responsible for life, health, and working conditions for Norwegianregistered ships and ships at Norwegian ports and is also in charge of active safety work, such as inspections regulated by international law. The Norwegian Coastal Administration is responsible for maritime infrastructure and maritime safety services, while the Labour Inspection Agency is responsible for working conditions in ports. Safety in the maritime

sector in Norway is regulated through the Ship Safety and Security Act, which came into existence in 2007. According to this act:

"The company has an overall duty to see to that the construction and operation of the ship is in accordance with the rules laid down in or pursuant to this Act, including that the master and other persons working on board comply with the legislation". (§6)

Section 58 in this act relates to the establishment of SMS (as required by the ISM code).


**4. Chitosan as a vector for gene therapy**

Cationic polymers, such as chitosan, are promising candidates for DNA transport in nonviral delivery systems (Kean & Thanou, 2010; Tong et al., 2009). Chitosan, a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), and has once been considered as an attractive gene transfer candidate for its superior biocompatibility, superior biodegradability and low cell toxicity. In recent years, with more researching methods involved, a more accurate and subtle view on the process of the entry of chitosan/DNA complexes into the cell nucleus has been developed. The enabling characteristics of Chitosan-DNA nanoparticles include biocompatibility, multiple ligand affinity, and a capacity of taking up large DNA fragments, while remaining small in size (Techaarpornkul et al., 2010). Chitosan and its derivatives, as favorable non-viral vectors involved in plasmid DNA delivery, have attracted attention in the field of siRNA delivery *in vitro* and *in* vivo (Andersen et al., 2009; Howard et al., 2009). Chitosan was once believed to be less effective than most other non-viral vectors because of its low stability and buffering capacity. However, recent technological advances in the chemical modification of chitosan have instituted improvements of its transfection efficiency without disturbing its biocompatibility and biodegradability. It has been demonstrated that transfection level is closely related to the molecular weight of polymers (Godbey et al., 1999). Chitosan (10-150 kDa), with a specific degree of deacetylation, allows maximum transgenic expression *in vitro*  (Lavertu et al., 2006). Another strategy for improving transfection is to take advantage of the mechanism of ligand-mediated uptake by cells to promote targeting and internalization, enhancing transfection efficiency. Ligand-mediated transfection has been shown to facilitate DNA internalization into cells via membrane receptors both *in vitro* and *in vivo.* Cell-specific ligand modification such as galactose, transferrin, folate and mannose can also effectively enhance the specificity of transfection through receptor-mediated endocytosis. Galactose ligand modification has been used to target HepG2 cells through the interaction with asialoglycoprotein receptors (ASGP-R) (Gao et al., 2003). A transferrin receptor is found on many mammalian cells, therefore it can be used as a universal ligand (Dautry-Varsat, 1986). Folate is not only over-expressed on macrophage surfaces, but is also over-expressed on many human cancer cell surfaces (Lee et al., 2006). Antigen presenting cells (APCs), the ideal targets of DNA vaccine, such as macrophages and immature dendritic cells are the target cells of mannose ligand (Kim et al., 2006). The specificity of these modifications can be demonstrated through ligand competitive inhibition experiments.

Chitosan-DNA/siRNA Nanoparticles for Gene Therapy 461

deacetylation in alkaline medium leads to the depolimerization (Domard & Rinaudo, 1983; Tolaimate et al., 2000). However it has been reported that chitin extracted from squid pens can be hydrolyzed under conditions that it allows obtaining chitosans of high molecular

Fig. 1. Chemical structure of chitin and chitosan. In the 2-amino-2-deoxy-d-glucopyranose

The homopolymer is a weak base with a pKa value of the D-glucosamine residue of about 6.3 and is therefore insoluble at neutral and alkaline pH values. In acidic mediums, the amine groups will be positively charged, conferring to the polysaccharide a high charge density. As in all polyelectrolytes, the dissociation constant of chitosan is not constant, but depends on the degree of dissociation at which it is determined. The pka value can be

Pka = pH + log [(1-α)/α] = pK0 –εΔψ(α)/kT Where Δψ is the difference in electrostatic potential between the surface of the polyion and the reference, α is the degree of dissociation, k is Boltzman's constant, T is the temperature and ε is the electron charge. Extrapolation of the pKa values to α = 1, where the polymer is uncharged and hence the electrostatic potential becomes zero, makes possible the value of the intrinsic dissociation constant of the ionizable groups, pK0, to be determined. The value obtained does not depend of the degree of *N*-acetylation, whereas the pKa value is dependent on this parameter, since the electrostatic potential will be varied depending of amount of the free amino groups. The pK0 value is called the intrinsic pKa of the chitosan. However chitosans of low molecular weight having degrees of deacetylation higher than 0.4 are also easily soluble in weakly acidic solvents such as

The physicochemical behavior in aqueous solution is highly dependent of pH and degree of acetylation and has received more attention only recently. Bertha et al. working on chitosans from 95 to 175kDa have recently determined the radius of gyration of chitosan (RG) (Bertha et al., 1998a; 2002b). The RG is an alternative measure of the size of the polymer chain and it can be measured by light scattering measurements. RG express the square mean radius of each one of the elements of the chain measured from its center of gravity. The study established the relationship between the molecular weight and radius of gyration (RG*)* of chitosan in aqueous solution, and the author indicated that chitosan behaved more like a Gaussian coil instead of the worm-like chain model found in common polyelectrolytes. At the same time the presence of *N*- acetyl groups on the chitosan backbone imparts hydrophobic properties. Schatz et al. (Schatz et al., 2003) have studied homogeneous series of chitosans with different degrees of acetylation and almost the same degree of

ring is shown the commonly used numbering for the carbon atoms.

calculated using the Katchalsky's equation (Roberts, 1992).

acetic acid and formic acid (Lee et al., 1995).

weight ( Tolaimate et al., 2003).

The low stability, low buffering capacity and low cell-specificity have also hindered its clinical applications. However, as a nature resource-based polysaccharide, chitosan has more functional groups that can be chemically modified than other cationic polymers, thus has many more potential chemical derivatives to overcome the deficiencies. Chitosan has been experimentally modified using hydrophilic, hydrophobic, pH-sensitive, thermosensitive and cell-specific ligand groups for enhancement of transfection efficiency (Ishii et al., 2001). The degree of deacetylation (DDA) and the molecular weight (MW) of chitosan or its derivatives, can affect the ultimate transfection efficiency. Most chitosan/DNA complexes are highly deacetylated (above 80%), because chitosan with a high degree of deacetylation exhibits an increased DNA binding efficacy (Kiang et al., 2004). Through chain entanglement, chitosan with a higher MW (longer chain length) can become more readily enmeshed with free DNA, once the initial electrostatic interaction has occurred. But it will also delay the disassociation of chitosan and DNA (Huang et al., 2005). Consequently, low MW chitosan requires a higher charge ratio to stably condense DNA for the same DDA, and a lower DDA requires a higher charge ratio to stably condense DNA at equal MW (Lavertu et al., 2006). The charge ratio for minimum complexation can be determined by agarose gel electrophoresis.


**Author details**

Sidonia Catalina Vrabie1 \*, Liliana Novac1 , Maria Magdalena Manolea1 , Lorena Anda Dijmarescu1 , Marius Novac2 and Mirela Anisoara Siminel3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Obstetrics and Gynecology, University of Medicine and Pharmacy, Craiova, Romania

2 Department of Anesthesiology and Intensive Care, University of Medicine and Pharmacy, Craiova, Romania

3 Department of Neonatology, University of Medicine and Pharmacy, Craiova, Romania


**1. Introduction**

For health and environmental considerations, the current community strongly demands to improve air quality. Environmental protection agencies around the world have introduced more and more stringent motor vehicle exhaust emission regulations on CO, nitrogen oxides (NOx), unburned hydrocarbons and particulate matter [1]. The commercial technology of pollutants elimination is to adopt efficient catalytic converters. Cu/SSZ-13 (SSZ-13 is one framework type code of chabazite zeolite), one of the most excellent NOx removal catalysts, has been commercially used in the exhaust after-treatment system of medium and heavy diesel vehicles [2–6].

In the past decade, numerical theoretical and experimental studies have revealed the pore structure, NH3-SCR catalytic active sites, high-temperature hydrothermal stability, sulfur poisoning mechanism and alkali metal poisoning mechanism of Cu/SSZ-13 [7–20]. Alkali metals, such as sodium (Na) and potassium (K) are present in conventional diesel fuel and biodiesel fuel, and have been implicated in internal injector deposits [21,22]. Besides, alkali metals also exist in automotive urea, such as diesel exhaust fluid (DEF) or AdBlue. Previous reports have shown that alkali metal ions are volatilized under hightemperature conditions and diffuse to the surface of the catalyst, causing the catalyst to be poisoned [23,24]. Fundamental researches have revealed the alkali metal poisoning mechanism of Cu/CHA catalysts [25,26]. Researchers suggest that alkali metal ions can destroy the active center Cu2+ and degrade the zeolite framework, causing irreversible deactivation. However, how to improve the tolerance of Cu/zeolite catalysts to alkali metals is rarely reported. Yan et al. reported that highly dispersed Cu4AlOx mixed oxides have a good alkali metal resistance [27]. However, the multiple Cu complex cannot fit in

**Citation:** Chen, Z.; Shen, M.; Wang, C.; Wang, J.; Wang, J.; Shen, G. Improvement of Alkali Metal Resistance for NH3-SCR Catalyst Cu/SSZ-13: Tune the Crystal Size. *Catalysts* **2021**, *11*, 979. https:// doi.org/10.3390/catal11080979

Academic Editor: Anker Degn Jensen

Received: 21 July 2021 Accepted: 16 August 2021 Published: 16 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the CHA's steric pore size and the inactive CuO clusters are easily formed in the CHA zeolites [28]. Du et al. compared the alkali resistance of conventional V2O5/WO3−TiO2 catalysts and Fe2O3/HY zeolite catalysts and found that the HY zeolites played an alkalibuffer role to retain the catalytic activity [29]. Zha et al. developed a hollandite Mn–Ti oxide promoted Cu-SAPO-34 (SAPO-34 is one framework type code of chabazite zeolite) catalysts (HMT@Cu-S). HMT layer can trap alkali metal ions to protect Cu/SAPO-34 [30]. The similarity of previous research lies in the use of outer shell materials to protect the inner structure. However, the synthesis of core–shell materials often requires fine control, and it is difficult to achieve industrial low-cost production. To meet industrial needs, we tried to develop a simple preparation process to obtain core–shell-like molecular sieves with good alkali metal resistance. In our previous study [31], it was found that different crystal sizes of the Cu/SAPO-34 can affect the spatial distribution of acid sites, which inspired us to adjust the acid sites on the outer surface of the molecular sieve by tuning the crystal size.

In this work, we report that Cu/SSZ-13 catalysts with different crystal sizes (0.4–2.3 µm) present distinct sodium metal ions resistance. Due to the similar deactivation effect of Na and K, we only choose Na to study the improvement of alkali metal resistance [25]. Na-poisoned Cu/SSZ-13 with crystal size of 2.3 µm showed much higher NH3-SCR activity than ones with a size of 0.4 µm. The distribution of acidity and Cu ions is characterized by H2-TPR, UV–vis, diethylamine-TPD, and pyridine-DRIFTs. By enlarging the crystal size of SSZ-13, the active center Cu ions are allowed to locate the subsurface or inner core, so that the outer layer of the zeolites buffer the foreign alkali metal poisoning. Adjusting the distribution of acid sites by modifying the crystal size provides a simple technical route to improve alkali metal poisoning.

#### **2. Results and Discussions**

The crystallization process of SSZ-13 zeolites is mainly divided into induction period and crystallization period [32]. During the induction period, the raw materials in the gel gradually gather to form crystal nuclei, and the crystal grows rapidly after entering the crystallization period. The crystal size of SSZ-13 can be affected by the crystallization time, crystallization temperature, gel pH and added amount of crystal seed. Through a series of trials, we found that the crystal size of SSZ-13 is most sensitive to the amount of seed crystal added. The as-prepared catalysts are visualized by SEM. As shown in Figure 1, the average crystal size of each sample is obtained based on the statistical results of ~100 grains. All samples present cubic shape and uniform particle size. The results show that the crystal sizes of the synthesized SSZ-13 are 0.4, 0.8, and 2.3 µm.

1 **Figure 1.** SEM images of Cu/SSZ-13 catalysts with different crystal sizes.


**3. Results and Discussion**

An overview of the chemical composition of almond tree biomass was conducted in order to describe the biomass potential of this species and how the selection of the vegetal material (variety and rootstock) and management practices can influence the properties of the obtained biofuel.

Table 2 presents the values of proximate analysis, elemental composition and gross calorific value of the studied samples. All variables, except the percentage of hydrogen, present standard kurtosis and standard skewness between −2 and +2, ANOVA analysis can be performed, as they all follow normal distributions. The average gross calorific value obtained in our analysis shows similar results to those obtained in almond hulls and shells by Nhuchhen [44] or by Fernández-González [19] or Yin [26] for biomass. The results are also close to those found for other *Prunus* species, such as *Prunus Avium* L. [45] or *Prunus armeniaca* L. [46]. As can be observed, the concentrations of C and H were 48.11% and 5.77%, respectively, which are the same as those observed by Jenkins [47] for almond residues. Percentages of C, H, N are in the range of published data for other biomass groups (C = 42–71%; H = 3–11%; N = 0.1–12%) [48]. Low concentration of N, as is the case, is a positive characteristic, as high N produces a negative impact on the environment due to nitrogen dioxide emissions [19]. The greatest disadvantage of biomass when it is used as fuel is its high moisture content, which is inversely correlated with its calorific value [49]. Almond trees presented an average moisture content (8.40%) much lower than the average for woody species (20%) [50], which therefore makes it convenient for energetic uses.

The dependence of the studied variables is shown in Table 3. It has been proved in other studies that moisture content and percentages of C and H have a high influence on gross calorific value [45,51]. However, in our study, we observed that the variables that mostly influence the gross calorific value are not the composition of C, H, N, but variety, rootstock and, above all, variety-rootstock interaction. We found that the variable that influenced the percentage of N the most was the treatment applied. This result is consistent with other research [52,53] that concludes that fertilizers and pesticide doses are highly important for some elements, such as the nitrogen content.

Given the obtained results, a variance analysis was carried out in order to compare the influence of the different factors analyzed in this study.


**1. Introduction**

592 Advances in Wavelet Theory and Their Applications in Engineering, Physics and Technology

Soucy, M. & Laurendeau, D. (1996). Multiresolution Surface Modeling Based on Hierarchical

Taubin, G., Guéziec, A., Horn, W., William, A. & Lazarus, F. (1998). Progressive Forest Split

Taubman, D. (2000). High Performance Scalable Image Compression with EBCOT. *IEEE*

Taubman, D. & Marcelin, M. (2001). *Jpeg2000: Image Compression Fundamentals, Standards and*

Wu, X. (1997). High-Order Context Modeling and Embedded Conditional Entropy Coding

1996), pp. 1-14, ISSN 10773142.

7149.

Orlando, Florida, USA, July 19-24, 1998.

*Practice*. Springer, ISBN<PHONE_NUMBER>197.

Triangulation. *Computer Vision and Image Understanding*. Vol. 63, No. 1, (January

Compression. *ACM SIGGRAPH, Proceedings of 25th International Conference on Computer Graphics and Interactive Techniques*, pp. 123-132, ISBN 0-89791-999-8,

*Transactions on Image Processing*. Vol. 9, No. 7, (July 2000), pp. 1158-1170, ISSN 1057-

of Wavelet Coefficients for Image Compression. *31st Asilomar Conference on Signals, Systems and Computers*, pp. 1378-1382, Pacific Grove, CA, November 2-5, 1997.


**Chapter 9 183**

A Multitiered Holistic Approach to Traffic Safety: Educating Children, Novice Teen Drivers and Parents, and Crash Investigators to Reduce Roadway Crashes - An Eight-Year Introspective Project *by Claudia M. Knezek, Susan Polirstok, Roxie James, Anthony Pittman and Gary Poedubicky*


*2.5. Cystic Kidney Disease*

Cystic kidney disease is one of the main renal ciliopathies [36,76]. Renal tubular cells detect fluid flow through cilia. In these cells, fluid flow increases Ca2+ uptake through calcium channels, such as polycystin 2 [77,78], and the consequent increase in the intracellular Ca2+ concentration inhibits adenylate cyclase 6 and suppresses cyclic adenosine monophosphate (cAMP) signaling. The dysfunction of cilia in renal tubular cells prevents the increase in the Ca2+ concentration and suppression of cAMP signaling in response to fluid flow, resulting in activation of protein kinase A [47,48]. In turn, protein kinase A activation increases fluid secretion through chloride channels and deregulates multiple cellular pathways, including proliferation, apoptosis, and the polarity of renal tubular cells, leading to renal cystogenesis [79].


**5. Optimal Design of WPHE for Ammonia Synthesis Column**

The mathematical model presented Section 3, validated in industrial conditions, can be used for the calculation of the optimal parameters of WPHE construction, as well as for WPHE optimal design based on plates of specific geometry. Here, the problem of WPHE optimisation consisting of finding the best construction parameters satisfying specified process conditions on heat transfer and pressure drop performance is considered. An approach discussed in [36] is used. The objective function is the heat transfer surface area of WPHE. There are a number of constraints that must be satisfied:

*Energies* **2020**, *13*, 2847

The estimation of the heat transfer performance of the WPHE is done through the determination of heat transfer effectiveness ε*T*. The total effectiveness of the heat exchanger with symmetric passes and countercurrent flow is determined by Equation (13) with the ratio of heat capacities flowrates *R* calculated by Equation (11). The value of effectiveness in one-pass ε*x*, required to satisfy the effectiveness of the whole WPHE, can be derived from Equation (13) as follows:

$$\varepsilon\_{X} = \left[ \left( \frac{1 - \varepsilon\_{T} \cdot \mathbf{R}}{1 - \varepsilon\_{T}} \right)^{\frac{1}{n}} - 1 \right] \cdot \left[ \left( \frac{1 - \varepsilon\_{T} \cdot \mathbf{R}}{1 - \varepsilon\_{T}} \right)^{\frac{1}{n}} - \mathbf{R} \right]^{-1} \tag{19}$$

The number of transfer units in one block of plates corresponding to one pass of crossflow WPHE from Equation (15):

$$\text{NTU}\_x^0 = -\frac{\ln(1 + 0.97 \cdot \text{R} \cdot \ln(1 - \varepsilon\_x))}{\text{R}} \tag{20}$$

It is required for fulfilment by WPHE of specified thermal process conditions. The number NTU*x* that can be obtained in one block of WPHE plates corresponding to one pass is according to the heat transfer ability of the block:

$$\text{NTU}\_{\text{x}} = \frac{\text{F}\_{\text{ax}} \cdot \text{U}}{\text{G}\_{2} \cdot \text{c}\_{p2}} \tag{21}$$

where F*ax* is the heat transfer area of the block of plates, m2.

Expressing F*ax* through the number of plates in one block and G2 through the number of channels and flow velocity in channel *w2* and channel cross-section area it is obtained:

$$\text{NTU}\_{\text{x}} = \frac{\text{2} \cdot \text{F}\_{\text{pl}} \cdot \text{U}}{c\_{p2} \cdot w\_2 \cdot \rho\_2 \cdot f\_{\text{cl}}} \tag{22}$$

where F*pl* is the heat transfer area of one plate, m2; ρ2 is the density of the cold fluid, kg/m3; *f* ch = *W*·*b* is the channel cross-section area, m2; *W* is the width of the channel, m.

The plate heat transfer surface area:

$$\mathbf{F}\_{pl} = \mathbf{L}\_{pl} \cdot \mathbf{W} \cdot \mathbf{F}\_{\mathbf{X}} \tag{23}$$

For the WPHE of ammonia synthesis column, the length of the plate L*pl* is equal to its width *W* and, from Equation (22), when the thermal constraint (iii) is completely satisfied (NTU*x* = NTU0*x*), Equation (24) follows:

$$\frac{\mathbf{I}\_{pl}}{b} = \frac{\mathbf{NTU}\_{\mathbf{x}}^{0} \cdot \mathbf{c}\_{p2} \cdot \mathbf{w}\_{2} \cdot \rho\_{2}}{\mathbf{2} \cdot \mathbf{U} \cdot \mathbf{F}\_{\mathbf{X}}} \tag{24}$$

Following the approach of [36], let us determine the plate length for the condition that pressure drop of the hot stream is strictly satisfied. From Equation (12), when the pressure drop ΔP1 is equal to the allowable pressure drop ΔP1 0,

$$\frac{\mathcal{L}\_{pl}}{b} = \frac{2}{\zeta\_1(w\_1)} \cdot \left(\frac{\Delta \mathcal{P}\_1^o \cdot 2}{\rho\_1 \cdot w\_1^2 \cdot n} - \zeta\_{\mathcal{D}Z1} \cdot \left(\frac{\mathcal{W}\_{\rm ch}}{\mathcal{W}\_{\rm emx}}\right)^2\right) \tag{25}$$

The flows velocities at the cold side (*w*2) and hot side (*w*1) are linked by Equation (26):

$$w\_2 = w\_1 \cdot \frac{\mathbf{G}\_2 \cdot \rho\_1}{\mathbf{G}\_1 \cdot \rho\_2} \tag{26}$$

When the hydraulic conditions by Equation (25) and thermal conditions by Equation (24) are satisfied simultaneously, the right sides of Equations (25) and (24) are equal. Accounting for Equation (26) for velocity in the hot channel, Equation (27) follows:

$$w\_1 = \sqrt{\frac{\Delta \mathbf{P}\_1^0}{n \cdot \rho\_1} \cdot \frac{1}{\frac{\zeta\_{\text{D} \text{T}\_1}{2} \cdot \left(\frac{\mathbf{W}\_{\text{ch}}}{\mathbf{W}\_{\text{ann}}}\right)^2 + \zeta\_1 (w\_1) \cdot \frac{\text{NTU}\_x^0 \mathbf{c}\_{p2} \cdot w\_1 \cdot \mathbf{C}\_2 \cdot \rho\_1}{8 \cdot \mathbf{U} (w\_2 w\_1) \cdot \mathbf{F}\_\mathbf{x} \cdot \mathbf{C}\_1}}}}\tag{27}$$

The variables ς and U at the right side of Equation (27) are the nonlinear functions of velocity *w*1 expressed by Equations (4)–(7). The solution of Equation (27) gives the value of *w*1 at which constraints (iii) and (iv) are satisfied as equalities and a corresponding value of overall heat transfer coefficient U is estimated. As a result, the value of WPHE heat transfer area for WPHE with *n* passes:

$$\mathbf{F}\_{\mathbf{d}} = \frac{\mathbf{NTU}\_{\mathbf{x}}^{0} \cdot \mathbf{G}\_{2} \cdot \mathbf{c}\_{p2}}{\mathbf{U}} \cdot n \tag{28}$$

The relations determining this objective function include nonlinear Equations and integer variable number of passes *n*. Besides, the numbers of plates and channels are also integers. To find the optimum of such function is the problem of Mixed-Integer Nonlinear Programming (MINLP). The numbers of plates and channels have rather big values and, on the first stage of finding the solution, can be treated as continues variables. The Equation (27) is a recurrent relation relative to velocity *w*1. Its solution by iterations gives the value of the velocity of a hot stream that, for a plate with specified geometrical parameters, strictly satisfies the thermal conditions and allowable pressure drop at the hot stream in a one-pass block of plates. When this velocity is obtained, the required plate length *Lpl* can be calculated by Equations (24) or (25). In case of a correct solution for *w*1, both are giving the same results. To satisfy the constraint (i), this plate length must be equal to the value specified for a given diameter of the column. The problem is solved by finding local optimums of plate spacing *bopt.i* at which constraint (i) for plate length is satisfied as equality. It is made for the series of increasing pass numbers *n*, starting from 2. The calculations are finished when *bopt.i* begin to increase. The value of *bopt.i* corresponding to a minimum of objective function F*a* and corresponding it passes number *n* are regarded as an optimal solution. After that, the number of plates in one block is rounded up, and rating design of WPHE performed. The method is implemented as a software for PC. The time of calculations for different conditions have not exceeded 200 s. The process of finding a solution is illustrated in the following case study.


4.2.1. Hydrogeochemical Facies

A Piper diagram can help to evaluate the geochemical relationships between different dissolved ions and dominant types of water chemistry in groundwater [38]. The TDS of shallow groundwater was 738.4–2532.6 mg/L [39]. The shallow groundwater was highly variable with HCO3 −, SO4 2−, Cl−, NO3 − as the main anions and Na+, Ca2+, Mg2+ as the main cations. The NO3 − concentration in the water samples was high and exceeded the quality standard for domestic drinking water, probably due to the inappropriate use of fertilizers making NO3 − enter the groundwater with rainfall and irrigation [40]. The HCO3-Cl-Mg-Ca type, SO4-HCO3-Na type and HCO3-Mg-Ca-Na type characterized the hydrogeochemical composition of shallow groundwater (Figure 3). The groundwater below the chromium slag pile (CSP) was SO4-HCO3-Na type. The reason was related to the production process of chromium salt, and the specific chemical reaction was 2Na2CrO4 + H2SO4 = Na2Cr2O7 + Na2SO4 + H2O, so SO4 2− and Na+ will enter the groundwater under the leaching effect of chromium slag. The downstream was basically uncontaminated, and the groundwater was HCO3-Mg-Ca-Na type. The groundwater of middle reaches and east of the chromium slag piles was HCO3-Cl-Mg-Ca type.

**Figure 3.** Piper diagram for shallow and deep groundwater. CSP represents chromium slag pile.

The TDS of deep groundwater in the site was 504–1198.26 mg/L. The anions were mainly HCO3 −, SO4 2−, Cl−, NO3 −, and the cations were mainly Na+, Ca2+, Mg2+. The NO3 − concentration in the deep groundwater was lower than that in the shallow groundwater, but still exceeded the quality standard of domestic drinking water. It may be that the shallow groundwater overflowed into the deep groundwater, resulting in abnormal NO3 − concentration in the deep groundwater. The deep hydrogeochemical composition was simpler than the shallow ones, and the groundwater was mainly HCO3-Cl-Mg-Ca type, HCO3-Na-Mg type and HCO3-SO4-Mg-Na-Ca type.


*Edited by Mark Wickham*

The knowledge management concept has emerged to serve as one of the critical inputs to the strategic management process, and a common factor underpinning competitive advantage. Over the concept's development, knowledge management research has focused on the processes that enable a firm to recognize sources of data, to transform data into useful information, to disseminate the information, and to develop strategies based on its insights. More recently, the development of the concept has begun to focus on the critical antecedents that enable these knowledge management processes to be implemented more effectively and efficiently. This research book serves to highlight some of the antecedents of effective knowledge management through empirical research done by researchers all around the globe.

Published in London, UK © 2019 IntechOpen © i3D\_VR / iStock

Current Issues in Knowledge Management

Current Issues in

Knowledge Management

*Edited by Mark Wickham*


**Physical Mechanisms of "Poisoning" the Living Organism by Heavy Metals**

G.P. Petrova *Lomonosov Moscow State University, Russia*

### **1. Introduction**

22 Environmental Monitoring

Tyler, C.R., Jobling, S. & Sumpter, J.P. (1998). Endocrine disruption in wildlife: a critical

United States-EPA (2007). Pharmaceuticals and personal care products in water, soil,

Zacharewski, T. (1997). *In vitro* bioassays for assessing estrogenic substances. *Environmental*

Zheng, W., Yates, S. & Bradford, S. (2008). Analysis of steroid hormones in a typical dairy waste disposal system. *Environmental Science & Technology* 42: 530-535.

review of the evidence. *Critical Reviews in Toxicology* 28: 319-361.

sediment, and biosolids by HPLC/MS/MS. *Method 1694*.

*Science & Technology* 31: 613-623.

The toxic affect of heavy metals over the living organisms is known to arouse from the alternation of the course for biological reactions in cells. One of such violations appears to be the process of supra-molecular structures formation, for example, the dipole protein nanoclusters in blood. This phenomenon can be well studied in the biological solutions, such as blood serum, or a widely adopted normal saline solution of albumin [1-3].

In the works devoted to problems of pollution of the surrounding environment and ecological monitoring, for today to heavy metals carry more than 40 metals of periodic system D.I. Mendeleyev with nuclear weight over 50 nuclear units: V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, Cd, Sn, Hg, Pb, Bi, etc. Thus the important role in categorize heavy metals is played by following conditions: their high toxicity for live organisms in rather low concentration, and also ability to bioaccumulation. Almost all metals getting under this definition (except for lead, mercury, cadmium and the bismuth which biological role currently is not clear), actively participate in biological processes, are a part some many enzymes. On N.Rejmersa's classification, heavy it is necessary to consider metals with density more than 8 g/cm3 .Thus, heavy metals concern Pb, Cu, Zn, Ni, Cd, Co, Sb, Sn, Bi, Hg.

The results of our investigations can to conclude that the process of cluster formation depends on the value of ionic radius metal.

In our works the interaction of some proteins – albumins, globulins, collagen, lisozym, collagenase, creatin cenase, pepsin with heavy metal ions like Cs, Rb, Cu, Cd, Pb in water solutions was studied.

Especial influence on some protein like albumins, globulins, collagen, lisozym and so on has the potassium. K+ ions presence in the protein solutions also induced appearance of dipole protein nano-clusters.

The appearance of cluster cans disturbance metabolic processes in the cells, membranes, tissue.

The interaction of proteins with ions of alkaline heavy metals like Cs+, Rb+, and Cu2+, Cd2+, Pb2+ in aqueous solutions was studied in our earlier works [4-9] with the Rayleigh-Debye light scattering (RDLS), the photon correlation spectroscopy (PCS) and the fluorescence polarization (FP) methods. It should be noted, that the nano-sized cluster formation process was also registered in some proteins and enzymes solutions like albumin, globulin, collagen, lysozyme, collagenase and so on in the presence of K+ ions which does not belong to the heavy metals group [8, 11].

Physical Mechanisms of "Poisoning" the Living Organism by Heavy Metals 25

attraction instead of Coulomb repulsion, because the proteins have abnormally high dipole moments (several hundred Debye uniques). So the protein molecules can go closely to each

Fig. 2. The scheme of interaction processes small and big ions with protein surface. (In the formulae , Epp – the energy of dipole-dipole interaction, p is the protein molecule

**3. Different optical methods of heavy metals interaction with biological**

The RDLS method is reliable for the determination of static parameters of the solid compounds in the suspension. The way how the light is scattered in the solution bears the information on the effective mass and molecular interaction coefficient of the particles. In case of the diluted solutions the measured experimental value of the Rayleigh scattering coefficient *R90* is related within Debye's theory to the mass of a macromolecule *М*, according

*0*is the incident beam wavelength, *n0* and *n* are the refractive indexes of the

2

*A*

*N*

,

*dn*

*n dc H*

2

dipole moment, l- the distance between macromolecules)

**3.1 Rayleigh-Debye light scattering (RDLS) method**

to the virial expansion of the osmotic pressure by concentration *c*:

1 2 ... *cH Bc R M* ,

**macromolecules investigations**

90

where

dissolvent and the solution.

other and forming aggregates – dipole clusters (see fig 2)

Fig. 1. Metals and its ionic radii

#### **2. Physical model**

It was found earlier that cluster formation depends on metal ion radius [2–4]. Interaction of these ions with a protein surface involves, as a rule, their hydrated shells. In cases where protein solutions contain small ions like Na+ (the ion radius equals 0.87 Å), dipole clusters are not formed, because sodium ions are located near the protein surface surrounded by water molecules and cannot bind directly with the negative charges on the protein.

$$E\_{pq} = \frac{q^2 p\_w^2}{12\pi\varepsilon r\_0^4} \frac{1}{kT} \dots$$

The energy of the ion and the water dipole molecule binding, determined by equation (q- is the charge of the ion, pw – water molecule dipole moment, -is dielectrical permeability of water, r0- the ionic radius), is inversely proportional to the fourth power of the ionic radius. In the heavy ions case, it may be of the same order or less than the heat energy kT, and the water shell cannot stay on ion surfaces. This is observed for ions with large radii, such as Cs+, Rb+, Cd+, Ce+, Pb 2+, and Eu 3+, as well as K+. In interacting with the protein surface directly, a metal ion with a large radius is bound more strongly to negatively charged groups on the protein and can form a Coulomb complex on a protein macromolecule with a common hydrated shell. In this case, the metal ions compensate completely for the local surface charge of the protein molecule [5].

The effective decrease of the protein surface charge that takes place as a result of strong binding of metal ions with a large radius and the macromolecule can lead to a situation where the main type of interaction between the protein molecules is a dipole–dipole

It was found earlier that cluster formation depends on metal ion radius [2–4]. Interaction of these ions with a protein surface involves, as a rule, their hydrated shells. In cases where protein solutions contain small ions like Na+ (the ion radius equals 0.87 Å), dipole clusters are not formed, because sodium ions are located near the protein surface surrounded by

water molecules and cannot bind directly with the negative charges on the protein.

*w pq q p E*

The energy of the ion and the water dipole molecule binding, determined by equation

(q- is the charge of the ion, pw – water molecule dipole moment,

completely for the local surface charge of the protein molecule [5].

*r kT* 2 2

permeability of water, r0- the ionic radius), is inversely proportional to the fourth power of the ionic radius. In the heavy ions case, it may be of the same order or less than the heat energy kT, and the water shell cannot stay on ion surfaces. This is observed for ions with large radii, such as Cs+, Rb+, Cd+, Ce+, Pb 2+, and Eu 3+, as well as K+. In interacting with the protein surface directly, a metal ion with a large radius is bound more strongly to negatively charged groups on the protein and can form a Coulomb complex on a protein macromolecule with a common hydrated shell. In this case, the metal ions compensate

The effective decrease of the protein surface charge that takes place as a result of strong binding of metal ions with a large radius and the macromolecule can lead to a situation where the main type of interaction between the protein molecules is a dipole–dipole

4 0

12 .

1

Fig. 1. Metals and its ionic radii

**2. Physical model**

attraction instead of Coulomb repulsion, because the proteins have abnormally high dipole moments (several hundred Debye uniques). So the protein molecules can go closely to each other and forming aggregates – dipole clusters (see fig 2)

Fig. 2. The scheme of interaction processes small and big ions with protein surface. (In the formulae , Epp – the energy of dipole-dipole interaction, p is the protein molecule dipole moment, l- the distance between macromolecules)

#### **3. Different optical methods of heavy metals interaction with biological macromolecules investigations**

#### **3.1 Rayleigh-Debye light scattering (RDLS) method**

The RDLS method is reliable for the determination of static parameters of the solid compounds in the suspension. The way how the light is scattered in the solution bears the information on the effective mass and molecular interaction coefficient of the particles. In case of the diluted solutions the measured experimental value of the Rayleigh scattering coefficient *R90* is related within Debye's theory to the mass of a macromolecule *М*, according to the virial expansion of the osmotic pressure by concentration *c*:

90 1 2 ... *cH Bc R M* , 2 2 2 0 4 2 *A dn n dc H N* ,

where *0*is the incident beam wavelength, *n0* and *n* are the refractive indexes of the dissolvent and the solution.

Physical Mechanisms of "Poisoning" the Living Organism by Heavy Metals 27

experimental autocorrelation function of the scattered light intensity relates to the

(1) 2 ( ) exp( ) *t g D*

Fig. 4 shows the dependences of translation diffusion coefficient on pH for the pure gamma-

3456789

pH

Fig. 4.Translation diffusion coefficient as function of pH for -Globulin water solutions with

The *Dt* value is twice less in the latter case when studied in the isoelectric point area of pH~6. It means that the mass of the particles in the solution with K+ ions is one order greater

> 0 11 ~ *cluster K protein*

where, Mprotein is the molecular mass of protein and Mcluster - the mass of scattering particle.

The fluorescence polarization (FP) method was used to determine the orientation correlation time *trot* of albumin in the solutions containing Pb2+ and Na+ ions. This parameter is based on the fluorescence polarization experimental data [6] and is calculated according to the

where *tfl* is the lifetime of the excited state. The latter proportion determines linear

, *rot*

,

*V M t kT kT*

,

3

*D M D M*

*q*

*h* is viscosity, *hr* - hydrodynamic radius of the particle. The normalized

,

> Globulin Globulin + KCl

In this formulae

where, *q* is wave-vector,

and without K+ ions

translational diffusion coefficient *Dt* as:

globulin solution (a) and the one containing K+ ions (b).

0

5

D\*10-8 см2

than that of the gamma-globulin molecule:

**3.3 Polarized fluorescence method**

0 0 11 11

dependence of the *trot* on the mass *M* of the particle.

*PP P t*

3

*fl rot*

*t*

Levshin-Perrin relation [7]:

/с

10

15

Fig. 3 presents data for pH-dependences of particle mass values obtained by RDLS method for the cases of the *Egg* albumin solution with Cesium (a), the bovine serum albumin (BSA) and the Gamma-globulin solutions with Potassium (b,c). All three graphs reveal the formation of large particles, one order heavier than the initial protein molecule. It should be noted that the maximum mass of nano-clusters in case of the K+ ions in the solutions relates to the physiological pH values.

Fig. 3. (a) pH-dependencies of scattered particle mass for Egg albumin in water solution in presence of Cs ions (2) ( = 0,00105 mol/l), (1) - Egg albumin in pure water solution. (b) pHdependences of scattering particle mass for albumin, , containing ions K+. (c) pHdependences of scattering particle mass for -globulin water solutions, containing ions K+.

#### **3.2 Photon-correlation spectroscopy (PCS)**

The PCS method was suggested to investigate the dynamic parameters of proteins in the aqueous solutions containing heavy metals [4, 5]. The translational diffusion coefficient *Dt* is described by the Stocks-Einstein-Debye formula as:

$$D\_t = \frac{kT}{6\pi\eta\eta\_h}$$

In this formulae *h* is viscosity, *hr* - hydrodynamic radius of the particle. The normalized experimental autocorrelation function of the scattered light intensity relates to the translational diffusion coefficient *Dt* as:

$$\mathcal{S}^{(1)}(\tau) = \exp(-D\_t \eta^2 \tau) \ ,$$

where, *q* is wave-vector, - correlation time.

26 Environmental Monitoring

Fig. 3 presents data for pH-dependences of particle mass values obtained by RDLS method for the cases of the *Egg* albumin solution with Cesium (a), the bovine serum albumin (BSA) and the Gamma-globulin solutions with Potassium (b,c). All three graphs reveal the formation of large particles, one order heavier than the initial protein molecule. It should be noted that the maximum mass of nano-clusters in case of the K+ ions in the solutions relates

(a) (b)

**3 4 5 6 7 8 9 10 11**

**pH**

(c) Fig. 3. (a) pH-dependencies of scattered particle mass for Egg albumin in water solution in presence of Cs ions (2) ( = 0,00105 mol/l), (1) - Egg albumin in pure water solution. (b) pH-

dependences of scattering particle mass for -globulin water solutions, containing ions K+.

The PCS method was suggested to investigate the dynamic parameters of proteins in the aqueous solutions containing heavy metals [4, 5]. The translational diffusion coefficient *Dt* is

6 *t*

*kT D r*

*h*

dependences of scattering particle mass for albumin, , containing ions K+. (c) pH-

**M\*105, g/mol**

**2 3 4 5 6 7 8 9 10**

**pH**

**M/M0**

 **M/M0**

to the physiological pH values.

**0**

**2**

**4**

**6**

**8**

**10**

**M \*106, g/mol**

**345678**

**0,0**

**5,0x105**

**3.2 Photon-correlation spectroscopy (PCS)**

described by the Stocks-Einstein-Debye formula as:

**1,0x106**

**M, g/mol**

**1,5x106**

**2,0x106**

**2,5x106**

**1 pH**

**2**

Fig. 4 shows the dependences of translation diffusion coefficient on pH for the pure gammaglobulin solution (a) and the one containing K+ ions (b).

Fig. 4.Translation diffusion coefficient as function of pH for -Globulin water solutions with and without K+ ions

The *Dt* value is twice less in the latter case when studied in the isoelectric point area of pH~6. It means that the mass of the particles in the solution with K+ ions is one order greater than that of the gamma-globulin molecule:

$$\left(\frac{D\_0}{D\_K}\right)^3 = 11 \sim \frac{M\_{cluster}}{M\_{protein}}\,\prime$$

where, Mprotein is the molecular mass of protein and Mcluster - the mass of scattering particle.

#### **3.3 Polarized fluorescence method**

The fluorescence polarization (FP) method was used to determine the orientation correlation time *trot* of albumin in the solutions containing Pb2+ and Na+ ions. This parameter is based on the fluorescence polarization experimental data [6] and is calculated according to the Levshin-Perrin relation [7]:

$$\frac{1}{P} = \frac{1}{P\_0} + \left(\frac{1}{P\_0} - \frac{1}{3}\right) \frac{t\_{\beta}}{t\_{rot}} \,\prime \qquad\qquad\qquad\qquad t\_{rot} = \frac{V\eta}{kT} = \frac{M\eta}{\rho kT} \,\prime \,\prime$$

where *tfl* is the lifetime of the excited state. The latter proportion determines linear dependence of the *trot* on the mass *M* of the particle.

Physical Mechanisms of "Poisoning" the Living Organism by Heavy Metals 29

Thus, the FP method confirms the formation of the nano-sized clusters in the protein

In this part the sorption process of ions with various radii on the serum blood protein surface during the nano-clusters formation stage was study. A number of static parameters were achieved by Rayleigh-Debye light scattering, including effective masses and molecular interaction coefficient of the particles in the proteins aqueous solution containing ions of Na+, K+ and Pb2+ at different ionic strength. It was found that the nano-cluster formation

**Metal Mass, a. u. Nuclear charge Ionic radius, Å Relative mass of cluster**

**0,0 1,0x10-3 2,0x10-3 3,0x10-3 4,0x10-3 5,0x10-3**

Fig. 7. Rayleigh scattering coefficient (R90) as function of ionic strength of albumin water

I mol/kg

**4. Sorption of the ions with various ionic radii on protein surface**

The following table represents the metal ions as studied in this investigation:

**1,6x10-6** pH=3,4

11 *Na*23 23 11 0,87 <2 19 *K*39 39 19 1,33 20-35 82 *Pb*207 207 82 1,2 >20

> pH=5,0 pH=7,0

solutions with presence of heavy metal ions.

**in the process of nano-clusters formation**

process depends on the ionic radius of the metal.

**0,0**

**2,0x10-7 4,0x10-7 6,0x10-7 8,0x10-7 1,0x10-6 1,2x10-6 1,4x10-6**

R90

solution containing Na+ ions.

**4.1 Results and discussion**

Table 1.

Fig. 5. pH-dependence of time rotation of albumin in the water solutions with Pb2+ and Na+ ions.

1. BSA 6,4·10-6M + Na+ 5,6·10-3 M

2. BSA 6,4·10-6M + Pb2+8,3·10-10M

3. BSA 6,4·10-6M + Pb2+1,7·10-7 M

4. BSA 6,4·10-6M + Pb2+6,3·10-5 M

As Fig. 5 shows the orientation correlation time increases along with the concentration of the heavy metal Pb2+ ions.

For comparison, fig 6 shows the plot of relative clusters mass depends on relative concentration - metal/protein for BSA solutions with potassium and lead ions.

Fig. 6. Relative clusters mass dependences on relative concentration - metal/protein for BSA solutions with potassium and lead ions.

Thus, the FP method confirms the formation of the nano-sized clusters in the protein solutions with presence of heavy metal ions.

### **4. Sorption of the ions with various ionic radii on protein surface in the process of nano-clusters formation**

In this part the sorption process of ions with various radii on the serum blood protein surface during the nano-clusters formation stage was study. A number of static parameters were achieved by Rayleigh-Debye light scattering, including effective masses and molecular interaction coefficient of the particles in the proteins aqueous solution containing ions of Na+, K+ and Pb2+ at different ionic strength. It was found that the nano-cluster formation process depends on the ionic radius of the metal.

#### **4.1 Results and discussion**

The following table represents the metal ions as studied in this investigation:

Table 1.

28 Environmental Monitoring

**2345678**

Fig. 5. pH-dependence of time rotation of albumin in the water solutions with Pb2+ and Na+

As Fig. 5 shows the orientation correlation time increases along with the concentration of the

For comparison, fig 6 shows the plot of relative clusters mass depends on relative

Fig. 6. Relative clusters mass dependences on relative concentration - metal/protein for BSA

concentration - metal/protein for BSA solutions with potassium and lead ions.

**pH**

4

3

2 1

**0**

1. BSA 6,4·10-6M + Na+ 5,6·10-3 M 2. BSA 6,4·10-6M + Pb2+8,3·10-10M 3. BSA 6,4·10-6M + Pb2+1,7·10-7 M 4. BSA 6,4·10-6M + Pb2+6,3·10-5 M

solutions with potassium and lead ions.

heavy metal Pb2+ ions.

ions.

**20**

**40**

**60**

**80 t rot, nс**

Fig. 7. Rayleigh scattering coefficient (R90) as function of ionic strength of albumin water solution containing Na+ ions.

Physical Mechanisms of "Poisoning" the Living Organism by Heavy Metals 31

 pH=3,4 pH=4,8 pH=7,1

Fig. 9. R90 as the function of ionic strength in albumin water solution containing K+ ions.

**0,0 5,0x10-4 1,0x10-3 1,5x10-3 2,0x10-3 2,5x10-3 3,0x10-3 3,5x10-3 4,0x10-3**

**I mol/kg**

**0,0 5,0x10-4 1,0x10-3 1,5x10-3 2,0x10-3 2,5x10-3 3,0x10-3**

**I mol/kg**

Fig. 10. Dependence of relative masses of scattering particles for BSA solution, containing K+

As Fig. 11 shows the former changes its sign and becomes negative when the latter reaches the values in the area of 10-15 mmol/l. This effect is due to the change in the type of molecular interaction which is caused by the increment of the Pb2+ ions concentration. In this case the Coulomb repulsion between protein macromolecules, when *B* is positive,

diminishes, the pure dipole attraction takes over, and *B* descends below zero.

**0**

**5**

**10**

**15**

MB/M0

at pH=7.

**20**

**25**

**30**

**35**

**0,0**

**5,0x10-6**

**1,0x10-5**

**1,5x10-5**

**2,0x10-5**

**R90**

**2,5x10-5**

**3,0x10-5**

The mentioned above metals were used to study the dependence of the Rayleigh scattering coefficient *R90* on the value of the ionic strength *I* in the aqueous solutions of albumin produced by "Sigma Inc." (USA).

Fig. 7 shows the dependence of *R90* on *I* for the solution with Na+ ions, whereas Fig.8 shows the relative masses of scattering particles dependence for this solution at pH=7.0 on *I*, which is the concentration of Na+ ions in this case.

Fig. 8. Scattering particles (MP) relative mass in albumin (M0) solution as a function of ionic strength Na+.

As follows from these graphs the presence of Na+ ions in this solution at higher ionic strength slightly increases the masses of the scattering particles. Compared to the mass of the albumin molecule the masses of these particles are less than twice heavier, approx. ~ 1,8. Probably, a number of protein molecules in the albumin solution with Na+ ions can form dimers.

Contrary to that the effect is absolutely different with K+ and Pb2+ ions in the albumin solution.

Fig. 9 shows the dependences of *R90* on ionic strength in the BSA solution, containing K+ ions for a number of pH values. The dependence of relative masses of scattering particles for this solution at pH=7 is shown on Fig.10.

In this case the value of the relative mass *Mcluster/Mprotein* ,which represents the mass ratio of the nano-sized cluster to the albumin molecule, lies in the area of 20-35 for the ionic strength around 2-3 mmol/l.

The concentration variations of the Pb2+ ions in the albumin solution leads to a dramatic decrease of the molecular interaction coefficient, which is the second virial coefficient *B* upon the increase of the ionic strength.

The mentioned above metals were used to study the dependence of the Rayleigh scattering coefficient *R90* on the value of the ionic strength *I* in the aqueous solutions of albumin

Fig. 7 shows the dependence of *R90* on *I* for the solution with Na+ ions, whereas Fig.8 shows the relative masses of scattering particles dependence for this solution at pH=7.0 on *I*, which

**5,0x10-4 1,0x10-3 1,5x10-3 2,0x10-3 2,5x10-3 3,0x10-3 3,5x10-3**

**I mol/kg**

Fig. 8. Scattering particles (MP) relative mass in albumin (M0) solution as a function of ionic

As follows from these graphs the presence of Na+ ions in this solution at higher ionic strength slightly increases the masses of the scattering particles. Compared to the mass of the albumin molecule the masses of these particles are less than twice heavier, approx. ~ 1,8. Probably, a number of protein molecules in the albumin solution with Na+ ions can form

Contrary to that the effect is absolutely different with K+ and Pb2+ ions in the albumin

Fig. 9 shows the dependences of *R90* on ionic strength in the BSA solution, containing K+ ions for a number of pH values. The dependence of relative masses of scattering particles for

In this case the value of the relative mass *Mcluster/Mprotein* ,which represents the mass ratio of the nano-sized cluster to the albumin molecule, lies in the area of 20-35 for the ionic strength

The concentration variations of the Pb2+ ions in the albumin solution leads to a dramatic decrease of the molecular interaction coefficient, which is the second virial coefficient *B*

produced by "Sigma Inc." (USA).

is the concentration of Na+ ions in this case.

**1,0 1,1 1,2 1,3 1,4 1,5 1,6 1,7 1,8 1,9**

this solution at pH=7 is shown on Fig.10.

upon the increase of the ionic strength.

**M**

strength Na+.

dimers.

solution.

around 2-3 mmol/l.

**P/M**

**0**

Fig. 9. R90 as the function of ionic strength in albumin water solution containing K+ ions.

Fig. 10. Dependence of relative masses of scattering particles for BSA solution, containing K+ at pH=7.

As Fig. 11 shows the former changes its sign and becomes negative when the latter reaches the values in the area of 10-15 mmol/l. This effect is due to the change in the type of molecular interaction which is caused by the increment of the Pb2+ ions concentration. In this case the Coulomb repulsion between protein macromolecules, when *B* is positive, diminishes, the pure dipole attraction takes over, and *B* descends below zero.

Physical Mechanisms of "Poisoning" the Living Organism by Heavy Metals 33

values in the range from 0,05 mmol/kg to 0,17 mol/kg relate to the process of monolayer

As graph data shows that the scattering particles masses are more than 20 times greater than the mass of the albumin molecule. It depicts the process of the formation of the larger particles which appear to be the nano-sized clusters generated by a number of the original macromolecules. With the presence of Pb2+ ions in the solution the cluster formation process occurs at the significantly smaller ionic strength values of 0,15 mmol/kg, as compared to the case of K+ ions of 1,5 mmol/kg. Nonetheless, the cluster formation process runs faster in case of Pb2+ ions although the generated particles appear to be lighter than in the case with

 The interaction of the metal ions with the charged surface of the protein in the solution is studied by the measurement of the light scattering coefficient along with the

 The dependence of masses of the scattering particles on the ionic strength and pH of the solution shows the Langmuir sorption process which leads upon the monolayer

The nano-sized clusters form as a result of the phase transition when the Coulomb

 The nano-cluster formation process in the protein solution depends on the ionic radii of metal. The clusters are formed in case of the solutions containing K+ and Pb2+ ions,

Cluster formation process can explain toxic influence of heavy metal ions at the very

The work was supported by the Russian Foundation for Fundamental Research, grant No.

I would like to thank my colleagues Yu.M. Petrusevich, K.V. Fedorova, M.A.Gurova, M.S. Ivanova, V.P. Khlapov, A.M. Makurenkov, I.A. Sergeeva, T.N. Tikhonova, E.A.Papish,

[1] Edsall J.T. et al. "Light Scattering in Solutions of Serum Albumin: effects of charge and

[4] Petrova G.P., Petrusevich Yu. M., Evseevicheva A.N. //General Physiology and

[6] Petrova G.P., Petrusevich Yu.M., Ten D.I.// Quantum Electronics, 32(10), p.897 (2002).

ionic strength" // J. of American Chem. Soc., 1950, V.72, P.4641. [2] P.Debye. Light scattering in solutions. Journal Аppl.Phys. 15, 338-349, 1944 [3] Scathard G., Batchelder A.C., Brown A. J. Am.Chem.Soc.68 2610 (1946)

repulsion forces diminish and the pure dipole attraction forces take over.

whereas the presence of Na+ ions in the solution reveals no effect.

formation which takes place until the Langmuir saturation is achieved.

K+ ions.

**5. Conclusions**

09-02-00438-a.

**7. References**

**6. Acknowledgements**

concentration variation of the former.

saturation to the dipole cluster formation.

small concentration on the living organisms.

*In memoriam of professor Yuriy M. Petrusevich (1935-2010).*

N.V.Sokol for taking part in these investigations.

Biophysics, V.17(2),Р.97,(1998).

[5] Petrova G.P., at al.// Proceedings of SPIE, V.4263, p.150, (2001),

Fig. 11. Dependence of B (the second virial coefficient) from ionic strength in albumin solution with Pb++ ions.

Fig. 12. Dependence of relative mass value from ionic strength of albumin solution with Pb++ ions (pH=7, 5)

Fig. 12 shows the dependence of the relative scattering particles mass on the ionic strength of the solution. The curve possesses a small slope rise of the relative mass. The ionic strength values in the range from 0,05 mmol/kg to 0,17 mol/kg relate to the process of monolayer formation which takes place until the Langmuir saturation is achieved.

As graph data shows that the scattering particles masses are more than 20 times greater than the mass of the albumin molecule. It depicts the process of the formation of the larger particles which appear to be the nano-sized clusters generated by a number of the original macromolecules. With the presence of Pb2+ ions in the solution the cluster formation process occurs at the significantly smaller ionic strength values of 0,15 mmol/kg, as compared to the case of K+ ions of 1,5 mmol/kg. Nonetheless, the cluster formation process runs faster in case of Pb2+ ions although the generated particles appear to be lighter than in the case with K+ ions.