A simple microplate-based method for the determination of α-amylase activity using the glucose assay kit (GOD method).
For the first time, a reliable, simple, rapid and high-throughput analytical method for the detection and quantification of α-amylase inhibitory activity using the glucose assay kit was developed. The new method facilitates rapid screening of a large number of samples, reduces labor, time and reagents and is also suitable for kinetic studies. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. The test is done in microtitre plates with a total volume of 260μL and an assay time of 40min including the pre-incubation steps. The new method is tested for linearity, sensitivity, precision, reproducibility and applicability. The new method is also compared with the most commonly used 3,5-dinitrosalicylic acid (DNSA) method for determining α-amylase activity.
Division of Nutritional Biochemistry, National Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka; Postgraduate Institute of Science, University of Peradeniya, Sri Lanka. Electronic address: frizliya@gmail.com.
Rizliya
R
Visvanathan
Division of Nutritional Biochemistry, National Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka. Electronic address: chatunii@gmail.com.
Chathuni
C
Jayathilake
Division of Nutritional Biochemistry, National Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka. Electronic address: ruvini@ifs.ac.lk.
Ruvini
R
Liyanage
D057075:Enzyme Assays / Q000379:methods*; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism*; D007700:Kinetics; D008320:Maltose / Q000378:metabolism*; D015203:Reproducibility of Results; D000516:alpha-Amylases / Q000032:analysis* / Q000378:metabolism*
D008320:Maltose; D005949:Glucose Oxidase; D000516:alpha-Amylases; D005947:Glucose
(+)-Catechin hydrate (PubChem CID: 9064); 3,5-Dinitrosalicylic acid (PubChem CID: 11873); Amylase inhibitors; Anhydrous dextrose (PubChem CID: 5793); DNSA; Gallic acid (PubChem CID: 370); Glucose oxidase; Maltose; Maltose monohydrate (PubChem CID: 16211336); Potassium sodium tartrate tetrahydrate (PubChem CID: 2724148); Rutin hydrate (PubChem CID: 45479757); Sodium phosphate dibasic (PubChem CID: 24203); Sodium phosphate monobasic (PubChem CID: 23672064); Starch soluble (PubChem CID: 439341); α-Amylase