2.9. Protein and Enzyme Analysis
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 [Amylase activity was measured in saliva by diluting the samples 1:100 in MILLI-Q and mixing them with alpha-amylase substrate consisting of 2-Chloro-4-nitrophenyl-α-D-maltotrioside (Apollo Scientific, Denton, UK, BITJ00020) as described earlier [Total protease activity (TPA) was measured using PEK-54 substrate ([FITC]-NIeKKKKVLPIQLNAATDK-[KDbc]) as described elsewhere [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 and 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 [