Detection of human urinary alpha-amylase encoded by the AMY2B gene using a fluorogenic substrate, FG5P

The existence of alpha-amylase (HXA) encoded by alpha-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, alpha-1,4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting alpha-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic alpha-amylase (HPA, 1.11) and human salivary alpha-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary alpha-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.