Effect of glycerol on protein acetylation by acetic anhydride

Antonio T. Fojo, Paul M. Reuben, Philip L. Whitney, William M. Awad

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

We investigated the basis for the previously unexplained stabilization of proteins by glycerol during reaction with acetic anhydride [S. Siegel and W. M. Awad, Jr. (1973) J. Biol. Chem. 248, 3233-3240]. Model studies showed that glycerol competes successfully for acetylation against protein hydroxyl groups. In contrast, amino groups are much more potent nucleophiles and their acetylation is not apparently affected. Since α-amino and phenolic pKa's did not change significantly in increasing glycerol concentrations, these findings are ascribed to glycerol's lower pKa value as compared to water, leading to the decreased acetylation of tyrosine, threonine, and serine hydroxyl groups in Pronase guanidine-stable chymoelastase. An additional mechanism is important and predominates in the protection against inactivation of bovine δ-chymotrypsin during acetylation and is explained by the recently described basis for protein stabilization in glycerol [K. Gekko and S. N. Timasheff (1981) Biochemistry 20, 4667-4676; 4677-4686]. Those studies demonstrated that glycerol increased the hydrophobicity of nonpolar residues, augmenting their tendency to be removed from protein surfaces. Therefore, the stabilization afforded by glycerol for chymotrysin is attributed in part to a favoring of the native folded state which forces the side chains of isoleucine-16 and valine-17 to be buried, increasing the apparent pKa of the α-amino group of isoleucine-16 as it forms the charge pair with the β-carboxyl group of aspartate-194. This conclusion was supported by stopped-flow analyses of the interaction of δ-chymotrypsin with proflavin in increasing concentrations of glycerol.

Original languageEnglish
Pages (from-to)43-50
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume240
Issue number1
DOIs
StatePublished - Jan 1 1985
Externally publishedYes

Fingerprint

Acetylation
Glycerol
Proteins
Stabilization
Isoleucine
Chymotrypsin
Hydroxyl Radical
Proflavine
Pronase
Nucleophiles
Biochemistry
acetic anhydride
Guanidine
Valine
Threonine
Hydrophobicity
Hydrophobic and Hydrophilic Interactions
Aspartic Acid
Serine
Tyrosine

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Effect of glycerol on protein acetylation by acetic anhydride. / Fojo, Antonio T.; Reuben, Paul M.; Whitney, Philip L.; Awad, William M.

In: Archives of Biochemistry and Biophysics, Vol. 240, No. 1, 01.01.1985, p. 43-50.

Research output: Contribution to journalArticle

Fojo, Antonio T. ; Reuben, Paul M. ; Whitney, Philip L. ; Awad, William M. / Effect of glycerol on protein acetylation by acetic anhydride. In: Archives of Biochemistry and Biophysics. 1985 ; Vol. 240, No. 1. pp. 43-50.
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