Determination of α-Amylase Using a Novel Blocked Substrate, 2-Chloro-4-Nitrophenyl 65-Azido-65-Deoxy-β-Maltopentaoside

Abstract

A novel blocked type substrate for use in a human α-amylase assay, 2-chloro-4-nitrophenyl 65-azido-65-deoxy-β-maltopentaoside (6-N3G5CNP), was synthesized by the chemical modification of 2-chloro-4-nitrophenyl-β-maltopentaoside (G5CNP) at the 6 position of the non-reducing-end with an azido group. This new substrate possesses the following properties:
1. Due to the modification of its 6-position OH group with the azido group, there was no hydrolysis of 6-N3G5CNP by the coupled enzyme glucoamylase.
2. Compared with G5CNP, 6-N3G5CNP has higher affinities to human pancreatic α-amylase (HPA) and human salivary α-amylase (HSA), with smaller Km values of 0.15mmol/l and 0.21mmol/l, respectively. Due to the greater specificity of its hydrolytic position, more than 96% of the product of hydrolysis of 6-N3G5CNP was G2CNP, compared with 81% when G5CNP was used as a substrate.
3. The reactivities to HPA and HSA were 97 and 99%, compared with 100% in each case for the non-blocked substrate G5CNP the reactivities of the two substrates were thus considered to be the same.
When used in an automatic analyzer, the α-amylase assay with 6-N3G5CNP yielded good results, confirming its useability. 6-N3G5CNP has all the properties required for use in the α-amylase assay. It will, therefore, be quite useful for the measurement of α-amylase activity in routine laboratories.