A method for simultaneous determination of the two possible products of acetohydroxy acid synthase

Natan Gollop; Ze'ev Barak; David M. Chipman

doi:10.1016/0003-2697(87)90054-6

A method for simultaneous determination of the two possible products of acetohydroxy acid synthase

Natan Gollop, Ze'ev Barak, David M. Chipman

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

23 Scopus citations

Abstract

A method for the simultaneous assay of 2-acetolactate and 2-aceto-2-hydroxybutyrate formation catalyzed by acetohydroxy acid synthase in the presence of its substrates pyruvate and 2-ketobutyrate is described. The method, appropriate for the study of the physiologcally and mechanistically significant competition between the two reactions, involves oxidative decarboxylation of the acetohydroxy acids to the corresponding 2,3-diketones, transfer of the volatile diketones to methanol, and gas chromatographic analysis with electron-capture detection. Oxidative decarboxylation by air requires catalytic activation, and addition of iron salts is crucial to the success of the method with purified enzymes.

Original language	English
Pages (from-to)	323-331
Number of pages	9
Journal	Analytical Biochemistry
Volume	160
Issue number	2
DOIs	https://doi.org/10.1016/0003-2697(87)90054-6
State	Published - 1 Feb 1987

Keywords

EC 4.1.3.18
acetohydroxybutyrate
acetolactate
acetolactate synthase
enzyme kinetics
gas chromatography (ECD)
oxidative decarboxylation

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/0003-2697(87)90054-6

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title = "A method for simultaneous determination of the two possible products of acetohydroxy acid synthase",

abstract = "A method for the simultaneous assay of 2-acetolactate and 2-aceto-2-hydroxybutyrate formation catalyzed by acetohydroxy acid synthase in the presence of its substrates pyruvate and 2-ketobutyrate is described. The method, appropriate for the study of the physiologcally and mechanistically significant competition between the two reactions, involves oxidative decarboxylation of the acetohydroxy acids to the corresponding 2,3-diketones, transfer of the volatile diketones to methanol, and gas chromatographic analysis with electron-capture detection. Oxidative decarboxylation by air requires catalytic activation, and addition of iron salts is crucial to the success of the method with purified enzymes.",

keywords = "EC 4.1.3.18, acetohydroxybutyrate, acetolactate, acetolactate synthase, enzyme kinetics, gas chromatography (ECD), oxidative decarboxylation",

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T1 - A method for simultaneous determination of the two possible products of acetohydroxy acid synthase

AU - Gollop, Natan

AU - Barak, Ze'ev

AU - Chipman, David M.

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Y1 - 1987/2/1

N2 - A method for the simultaneous assay of 2-acetolactate and 2-aceto-2-hydroxybutyrate formation catalyzed by acetohydroxy acid synthase in the presence of its substrates pyruvate and 2-ketobutyrate is described. The method, appropriate for the study of the physiologcally and mechanistically significant competition between the two reactions, involves oxidative decarboxylation of the acetohydroxy acids to the corresponding 2,3-diketones, transfer of the volatile diketones to methanol, and gas chromatographic analysis with electron-capture detection. Oxidative decarboxylation by air requires catalytic activation, and addition of iron salts is crucial to the success of the method with purified enzymes.

AB - A method for the simultaneous assay of 2-acetolactate and 2-aceto-2-hydroxybutyrate formation catalyzed by acetohydroxy acid synthase in the presence of its substrates pyruvate and 2-ketobutyrate is described. The method, appropriate for the study of the physiologcally and mechanistically significant competition between the two reactions, involves oxidative decarboxylation of the acetohydroxy acids to the corresponding 2,3-diketones, transfer of the volatile diketones to methanol, and gas chromatographic analysis with electron-capture detection. Oxidative decarboxylation by air requires catalytic activation, and addition of iron salts is crucial to the success of the method with purified enzymes.

KW - EC 4.1.3.18

KW - acetohydroxybutyrate

KW - acetolactate

KW - acetolactate synthase

KW - enzyme kinetics

KW - gas chromatography (ECD)

KW - oxidative decarboxylation

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DO - 10.1016/0003-2697(87)90054-6

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SN - 0003-2697

VL - 160

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JO - Analytical Biochemistry

JF - Analytical Biochemistry

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ER -

A method for simultaneous determination of the two possible products of acetohydroxy acid synthase

Abstract

Keywords

ASJC Scopus subject areas

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