title: Simple ITC method for activity and inhibition studies on human salivary α-amylase.
abstract: 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 α-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 KM 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-α-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 μM, respectively) and increased in parallel with the degree of polymerisation of substrates.
authors.affiliation: a Department of Inorganic and Analytical Chemistry , University of Debrecen , Debrecen , Hungary.
authors.forename: Gábor
authors.initials: G
authors.lastname: Lehoczki
authors.affiliation: a Department of Inorganic and Analytical Chemistry , University of Debrecen , Debrecen , Hungary.
authors.forename: Kármen
authors.initials: K
authors.lastname: Szabó
authors.affiliation: a Department of Inorganic and Analytical Chemistry , University of Debrecen , Debrecen , Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Takács
authors.affiliation: a Department of Inorganic and Analytical Chemistry , University of Debrecen , Debrecen , Hungary.
authors.forename: Lili
authors.initials: L
authors.lastname: Kandra
authors.affiliation: a Department of Inorganic and Analytical Chemistry , University of Debrecen , Debrecen , Hungary.
authors.forename: Gyöngyi
authors.initials: G
authors.lastname: Gyémánt
mesh_terms: D002151:Calorimetry / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D012463:Saliva / Q000201:enzymology*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000516:alpha-Amylases
keywords: Enzyme kinetics; maltooligosaccharides; method description; microcalorimetry