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

Gábor Lehoczki, Kármen Szabó, István Takács, L. Kandra, G. Gyémánt

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1-6
Number of pages6
JournalJournal of Enzyme Inhibition and Medicinal Chemistry
DOIs
Publication statusAccepted/In press - Apr 5 2016

Fingerprint

Calorimetry
Amylases
Starch
Acarbose
Spectrophotometry
Polymerization
Inhibitory Concentration 50
Hydrolysis
High Pressure Liquid Chromatography
Enzymes

Keywords

  • Enzyme kinetics
  • maltooligosaccharides
  • method description
  • microcalorimetry

ASJC Scopus subject areas

  • Drug Discovery
  • Pharmacology

Cite this

Simple ITC method for activity and inhibition studies on human salivary α-amylase. / Lehoczki, Gábor; Szabó, Kármen; Takács, István; Kandra, L.; Gyémánt, G.

In: Journal of Enzyme Inhibition and Medicinal Chemistry, 05.04.2016, p. 1-6.

Research output: Contribution to journalArticle

@article{e6d189f2de1e4699bca32b776e9eb638,
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.",
keywords = "Enzyme kinetics, maltooligosaccharides, method description, microcalorimetry",
author = "G{\'a}bor Lehoczki and K{\'a}rmen Szab{\'o} and Istv{\'a}n Tak{\'a}cs and L. Kandra and G. Gy{\'e}m{\'a}nt",
year = "2016",
month = "4",
day = "5",
doi = "10.3109/14756366.2016.1161619",
language = "English",
pages = "1--6",
journal = "Journal of Enzyme Inhibition and Medicinal Chemistry",
issn = "1475-6366",
publisher = "Informa Healthcare",

}

TY - JOUR

T1 - Simple ITC method for activity and inhibition studies on human salivary α-amylase

AU - Lehoczki, Gábor

AU - Szabó, Kármen

AU - Takács, István

AU - Kandra, L.

AU - Gyémánt, G.

PY - 2016/4/5

Y1 - 2016/4/5

N2 - 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.

AB - 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.

KW - Enzyme kinetics

KW - maltooligosaccharides

KW - method description

KW - microcalorimetry

UR - http://www.scopus.com/inward/record.url?scp=84964057558&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84964057558&partnerID=8YFLogxK

U2 - 10.3109/14756366.2016.1161619

DO - 10.3109/14756366.2016.1161619

M3 - Article

C2 - 27052104

AN - SCOPUS:84964057558

SP - 1

EP - 6

JO - Journal of Enzyme Inhibition and Medicinal Chemistry

JF - Journal of Enzyme Inhibition and Medicinal Chemistry

SN - 1475-6366

ER -