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Reaction of low molecular weight aminothiols with o-phthalaldehyde

Authors
  • Puri, Rajinder N.
  • Roskoski, Robert Jr.
Type
Published Article
Journal
Analytical Biochemistry
Publication Date
Jan 01, 1988
Volume
173
Issue
1
Pages
26–32
Identifiers
DOI: 10.1016/0003-2697(88)90153-4
Source
Elsevier
Keywords
License
Unknown

Abstract

o-Phthalaldehyde has been recently shown to be a useful reagent for chemical modification of cyclic nucleotide dependent protein kinases, hexokinase, and fructose-1,6-bisphosphatase. It reacts covalently with closely spaced (∼3 Å) sulfhydryl and ϵ-amino functions of cysteine and lysine residues, respectively, of these enzymes to yield fluorescent isoindole derivatives. We have found the reagent to be equally useful to investigate the degree of reactivity of sulfhydryl and amino functions in substances that do not possess enzymatic activity, e.g., glutathione, homocysteine, and cysteine. The kinetics of the reaction of nonenzymatic aminothiols with o-phthalaldehyde can be followed rapidly and conveniently by continuously monitoring the increase in relative fluorescence of the isoindole derivatives. The fluorescence emission maxima of the o-phthalaldehyde adducts can be used to compute molar transition energies that provide qualitative but useful information concerning the degree of polarity of microenvironment of the sulfhydryl and amino functions participating in isoindole formation. The kinetic and spectral data obtained from the reaction between o-phthalaldehyde and nonenzymatic low molecular weight aminothiols may be helpful in comparing the reactivities of the sulfhydryl and amino functions in enzymes.

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