Simple ITC method for activity and inhibition studies on human salivary alpha-amylase

摘要

Isothermal titration calorimetry (ITC) has an increasing significance in enzyme kinetic studies owing to its general applicability and sensitivity. In the present work, we aimed at developing a simple ITC-based screening procedure for the measurement of human salivary alpha-amylase (HSA) activity. Reaction of two substrates was studied with three independent methods (ITC, HPLC and spectrophotometry). ITC experiments were made using free and chromophore-containing maltooligomers of different length as substrates. Detailed studies revealed that maltoheptaose or longer oligomers could model properly starch and the presence of aromatic chromophore group did not affect the K-M values considerably. It is the first time, when ITC was used to investigate of HSA-catalysed hydrolysis of different substrates (2-chloro-4-nitrophenyl-4-O-alpha-D-galactopyranosyl-maltoside, maltoheptaose and starch) in the presence of acarbose inhibitor. All measured IC50 values are in micromolar range (0.9, 18.6 and 29.0 mu M, respectively) and increased in parallel with the degree of polymerisation of substrates.