Protein buffering in model systems and in whole human saliva

Abstract

The aim of this study was to quantify the buffer attributes (value, power, range and optimum) of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and alpha-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16%) between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate) staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s) belonging to the protein buffer system of human saliva.

Figure 1

Titration curves with 150 pH measurements per curve of (a) 5 mM di-hydrogenphosphate, (b) 10 mM hydrogencarbonate, (c) 10 mM hydrogencarbonate plus 5 mM di-hydrogenphosphate, (d) 10 µM (0.1%) amyloglucosidase, 340 µM (0.5%) lysozyme, 10 mM hydrogencarbonate and 5 mM di-hydrogenphosphate (model system I), (e) 10 µM (0.1%) amyloglucosidase, 40 µM (0.2%) α-amylase, 10 mM hydrogencarbonate and 5 mM di-hydrogenphosphate (model system II) and (f) deionized water. The calculated buffer power is indicated in µmol per 10 ml of the analytes, in the internal scale.

Figure 2

Titration curves with 86 pH measurements per curve of (a) 340 µM (0.5%) lysozyme in water, (b) 10 µM (0.1%) amyloglucosidase in water, (c) 10 µM (0.1%) amyloglucosidase plus 340 µM (0.5%) lysozyme in water, (d) 40 µM (0.2%) α-amylase and (e) 10 µM (0.1%) amyloglucosidase, 40 µM (0.2%) α-amylase.

Figure 3

Titration curves with 80 pH measurements per curve of amyloglucosidase in concentrations (a) 10 µM (0.1%), (b) 20 µM (0.2%) and (c) 50 µM and α-amylase in concentrations (d) 20 µM (0.1%), (e) 40 µM (0.2%) and (f) 100 µM (0.5%).

Figure 4

Titration curves with 80 pH measurements per curve of purified salivary protein from 10 ml saliva. Saliva samples were taken at (a) 9:00 am, (b) 13:00 and (c) 17:00. Next to the titration curves the corresponding electropherograms sections containing proteins from 50 to 110 kDa are shown. Proteins were visualized by modified ruthenium (ii) tris bathophenantroline staining.

Figure 5

Panel A: Titration curves with 150 pH measurements per curve of (a) human saliva, (b) 10 µM (0.1%) amyloglucosidase, 340 µM (0.5%) lysozyme, 10 mM hydrogencarbonate and 5 mM di-hydrogenphosphate (model system I) and (c) 10 µM (0.1%) amyloglucosidase, 40 µM (0.2%) α-amylase, 10 mM hydrogencarbonate and 5 mM di-hydrogenphosphate (model system II). Panel B: Titration curve with 150 pH measurements per curve of (a) titration curve with 150 averaged pH measurements (5 per pH measurement point) of 5 male subjects with standard deviations indicated by grey bars. (b) 10 µM (0.1%) amyloglucosidase, 340 µM (0.5%) lysozyme, 10 mM hydrogencarbonate and 5 mM di-hydrogenphosphate, (c) 10 µM (0.1%) amyloglucosidase, 40 µM (0.2%) α-amylase, 10 mM hydrogencarbonate and 5 mM di-hydrogenphosphate (model system II).