2.8. Protein and Enzyme Analysis
N′
ALDRICH
Saliva samples were centrifuged at 4 °C for 10 min at 10,000× Per the manufacturer’s instructions, the total protein concentration was analyzed as described [To measure the amylase activity, saliva samples were diluted 1:100 in MILLI-Q before testing. Next, 10 µL of the diluted saliva samples were mixed with 90 µL of a diluted alpha-amylase substrate consisting of 2-CHLORO-4-NITROPHENYL ALPHA-D-MALTOTRIOSIDE (Apollo Scientific, Denton, UK, BITJ00020) in assay buffer: 100 nm MES, 350 nM NaCl, 6 nM Calcium Acetate, 900 nM Potassium Thiocyanate, 0.1% with a final concentration of 1.8 nM Sodium Azide. Absorbance was measured at 450 nm for 15 min with 1 min intervals on a plate reader. For every run, 10 µL of an alpha-amylase standard of 1 U/mL (Sigma Aldrich, Saint Louis, MO, USA, 1 U/mL) mixed with 90 µL of the diluted alpha-amylase substrate was used as a positive control and to calculate U/mL of samples.Total protease activity (TPA) was measured using PEK-54 substrate ([FITC]-NIeKKKKVLPIQLNAATDK-[KDbc]) with a working solution of 32 μM diluted in TBS. Next, 50 µL of saliva and 50 μL of PEK-54 substrate were added to a black 96-well plate (non-binding), resulting in a final PEK-54 substrate concentration of 16 μM. Reaction mixes were placed in a fluorescence microplate reader with a 485 nm excitation filter and 530 nm emission filter (gain 800), and fluorescence was measured for approximately 1 h at 37 °C using 5 min scanning intervals. Proteolytic activity was expressed as the increase in fluorescence per min (F/min).To measure the chitinase activity, 50 µL 4-Methylumbelliferyl b-D-N, N′, N″-triacetylchitotriose substrate with a final concentration of 12.7 µM was mixed with 50 µL saliva in a black 96-well plate (non-binding). In every plate, a chitinase control enzyme with a concentration of 0.001 mg/mL was used as a positive control, while a substrate without samples or assay buffer was used as a negative control. Reaction mixes were placed in the fluorescence microplate reader with a 360 nm excitation filter and 450 nm emission filter, and fluorescence was measured for approximately 1 h at 37 °C using 5 min intervals.Albumin was measured as previously described [