exposure of ultraviolet ( uv ) irradiation to the skin causes acute and chronic detrimental cutaneous effects , which may result in photocarcinogenesis [ 17 ] .
native human melanin includes eumelanin and pheomelanin that contains sulfur , and eumelanin has been found in almost every type of human skin [ 8 , 9 ] . in the skin , melanin synthesized in melanocytes located in the basal layer and hair bulbs transfers to keratinocytes .
melanin in keratinocytes acts as a photoprotector through body coloration and scavenging reactive oxygen species [ 1015 ] . in spite of photoprotective role of melanin , there are many cosmetics developed to prevent melanin formation in the skin because of aesthetic satisfaction by whitening ability . of these ,
inhibitor of tyrosinase , which is a pivotal enzyme for melanin synthesis , has become major ingredient of cosmetics [ 1721 ] .
tyrosinase is an enzyme which catalyzes the biological conversion of tyrosine to dopaquinone with dioxygen at the dinuclear copper active site under physiological conditions [ 2224 ] .
other than oxidative catalysis of substrates by tyrosinase , a few studies on radical formation by tyrosinase have been reported .
for instance , it was reported that tyrosinase - dependent activation of hydroxybenzenes formed reactive compounds including free radical , and some radicals were produced during the tyrosinase reaction and dopa - melanin formation . however , in these few studies , radical species have not been determined .
we have studied the tyrosine - tyrosinase reaction in terms of melanin formation and ros scavenging ability of melanin . in a battery of studies
, we found radical formation through the enzyme reaction , and discuss the formation mechanism in this paper .
reagents were purchased from the following sources : l - tyrosine , phosphate buffer solution ( pb , ph  6.5 ) and catalase ( from bovine liver ) from wako pure chemicals ( osaka , japan ) ; tyrosinase ( from mushroom ) , and superoxide dismutase ( sod from bovine erythrocytes ) from sigma - aldrich corp .
( st . louis , mo ) ; 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo ) from labotec ( tokyo , japan ) ; deuterium water ( d2o ) from tokyo chemical industry co. , ltd ( tokyo , japan ) .
then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of 0.2  m pb . dmpo was dissolved in ultrapure water to be 4.5  m. the reaction mixture was prepared to contain different activity of tyrosinase , 20  l of 4.5  m dmpo , 60  l of 1  mm tyrosine and 0.2  m pb which was added to adjust a total volume of 200  l . immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 45  s. the measurement conditions of esr ( jes - fa-100 , jeol , tokyo , japan ) were as follows : field sweep , 330.80340.80  mt ; field modulation frequency , 100  khz ; filed modulation width , 0.07  mt ; amplitude , 400 ; sweep time , 1  min ; time constant , 0.1  s ; microwave frequency , 9.430  ghz ; microwave power , 45  mw .
then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of ultrapure water or d2o .
tyrosinase was dissolved in ultrapure water to be 100  u/l . immediately before the measurement tyrosinase was diluted to be 10  u/l with ultrapure water or d2o .
dmpo ( 8.9  m ) was diluted to be 4.5  m with ultrapure water or d2o .
the reaction mixture was prepared to contain 100  l of d2o , 20  l of 10  u/l tyrosinase ( 90% d2o solution ) , 20  l of 4.5  m dmpo ( 50% d2o solution ) , and 60  l of 1  mm tyrosine ( 99% d2o solution ) .
immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 60  s. as a control , the reaction mixture was prepared to contain 100  l of h2o ( ultrapure water ) , 20  l of 10  u/l tyrosinase dissolved in h2o , 20  l of 4.5  m dmpo dissolved in h2o , and 60  l of 1  mm tyrosine dissolved in h2o , and was similarly subjected to esr measurement .
reagents were purchased from the following sources : l - tyrosine , phosphate buffer solution ( pb , ph  6.5 ) and catalase ( from bovine liver ) from wako pure chemicals ( osaka , japan ) ; tyrosinase ( from mushroom ) , and superoxide dismutase ( sod from bovine erythrocytes ) from sigma - aldrich corp .
( st . louis , mo ) ; 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo ) from labotec ( tokyo , japan ) ; deuterium water ( d2o ) from tokyo chemical industry co. , ltd ( tokyo , japan ) .
then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of 0.2  m pb . dmpo was dissolved in ultrapure water to be 4.5  m. the reaction mixture was prepared to contain different activity of tyrosinase , 20  l of 4.5  m dmpo , 60  l of 1  mm tyrosine and 0.2  m pb which was added to adjust a total volume of 200  l . immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 45  s. the measurement conditions of esr ( jes - fa-100 , jeol , tokyo , japan ) were as follows : field sweep , 330.80340.80  mt ; field modulation frequency , 100  khz ; filed modulation width , 0.07  mt ; amplitude , 400 ; sweep time , 1  min ; time constant , 0.1  s ; microwave frequency , 9.430  ghz ; microwave power , 45  mw .
then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of ultrapure water or d2o .
tyrosinase was dissolved in ultrapure water to be 100  u/l . immediately before the measurement tyrosinase was diluted to be 10  u/l with ultrapure water or d2o .
dmpo ( 8.9  m ) was diluted to be 4.5  m with ultrapure water or d2o .
the reaction mixture was prepared to contain 100  l of d2o , 20  l of 10  u/l tyrosinase ( 90% d2o solution ) , 20  l of 4.5  m dmpo ( 50% d2o solution ) , and 60  l of 1  mm tyrosine ( 99% d2o solution ) .
immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 60  s. as a control , the reaction mixture was prepared to contain 100  l of h2o ( ultrapure water ) , 20  l of 10  u/l tyrosinase dissolved in h2o , 20  l of 4.5  m dmpo dissolved in h2o , and 60  l of 1  mm tyrosine dissolved in h2o , and was similarly subjected to esr measurement .
representative esr spectra obtained from tyrosine - tyrosinase reaction with different activity of tyrosinase are summarized in fig .  1
. each spectrum consists of quartet segments ( intensity ratio , 1:2:2:1 ) and triplet of triplet segments ( intensity ratio , 1:1:2:1:2:1:2:1:1 ) .
the former segment was assigned to dmpo - oh , a spin adduct derived from oh ( hyperfine coupling constant , an  =  1.49 ; ah  = 1.49  mt ) .
the latter segment was assigned to dmpo - h , a spin adduct derived from h ( hyperfine coupling constant , an  =  1.63 ; ah  =  2.25  mt ) as reported in a previous study .
the ratio of dmpo - h and dmpo - oh generated in the tyrosine - tyrosinase reaction was approximately 1:2 . to examine
if o2 and h2o2 are involved in a downstream of the generation process of h and oh , esr signals of dmpo - h and dmpo - oh generated by the tyrosine - tyrosinase reaction were analyzed in the presence of sod , a scavenger for o2 , and catalase , a cleaving enzyme for h2o2 .
neither sod ( 1  u/l ) nor catalase ( 1  u/l ) affected the amounts of dmpo - h and dmpo - oh ( data not shown ) , suggesting that neither o2 nor h2o2 is involved in the generation process of h and oh . then to further examine if h2o is a source of h and oh , esr signals of dmpo - h and dmpo - oh generated by the tyrosine - tyrosinase reaction where d2o was used as a major solvent ( 94% d2o solution ) were compared with those where h2o was used as a solvent . as shown in fig .  2 ,
the amount of dmpo - h was reduced by 70% , suggesting that approximately 70% of h is derived from h2o .
thus , we assume that the electron derived from the ligand of tyrosinase reacts with h2o or h to form h. that is , h2o  +  e 
oh  +  h or h  +  e  h. on the other hand , the amount of dmpo - oh was not affected by the replacement of h2o to d2o , suggesting that oh was derived from the catalytic reaction of tyrosine to dopaquinone by tyrosinase . since catalase had no effect on the generation of two radicals as described above , fenton - like reaction in which h2o2 reacts with transient metal copper in the structures of tyrosinase and its intermediates
is least likely involved in the generation mechanism of oh . as reported in the previous study on dinuclear copper complexes , a rather stable dicopper - peroxide intermediate in aqueous solution decays through an internal electron transfer from the ligand to produce oh . since tyrosinase is an enzyme which contains dinuclear copper ions at the active site , [ 2224 ] , dicopper - peroxide intermediates formed during the catalytic process of tyrosine to dopaquinone possibly decay to produce oh through an internal electron transfer from the ligand .
the proposed mechanism by which h and oh are generated in tyrosine - tyrosinase reaction is illustrated in fig . 
although tyrosine - tyrosinase reaction is a key step of melanin formation as a photoprotection , it also produces not only h but oh which might be a causative factor of oxidative damage of the skin .
since there are many cosmetics developed for whitening ability by inhibiting tyrosine - tyrosinase reaction , our study revealed that they also might contribute to alleviate the oxidative damage of the skin by inhibiting oh generation via the enzyme reaction .
 alleviated melanin formation in the skin through inhibition of tyrosine - tyrosinase reaction is one of the major targets of cosmetics for whitening ability . 
 since melanin has a pivotal role for photoprotection , there are pros and cons of inhibition of melanin formation . 
 this study applying electron spin resonance ( esr)-spin trapping method revealed that h and oh are generated through tyrosine - tyrosinase reaction . when deuterium water was used instead of h2o , the signal of 5,5-dimethyl-1-pyrroline n - oxide ( dmpo)-h ( a spin adduct of dmpo and h ) greatly decreased , whilst dmpo - oh ( a spin adduct of dmpo and oh ) did not . 
 thus , it is suggested that h was derived from h2o , and oh through oxidative catalytic process of tyrosine to dopaquinone . 
 our study suggests that tyrosinase inhibitors might contribute to alleviate the oxidative damage of the skin by inhibiting oh generation via the enzyme reaction . 
Introduction
Materials and Methods
Test materials and reagents
Electron spin resonance (ESR)-spin trapping determinations of ROS generated by tyrosine-tyrosinase reaction
Deuterium water (D
Results and Discussion