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Journal of Solid-Phase Biochemistry

, Volume 3, Issue 1, pp 57–70 | Cite as

Heterogeneity in the binding of chymotrypsin and related proteins to affinity chromatographic media

  • George Tomlinson
  • W. Charles Schellenberg
  • T. Viswanatha
Article
  • 22 Downloads

Abstract

The binding of a series of related proteins, namely, α-chymotrypsin, chymotrypsinogen A, DIP-chymotrypsin, and TPCK-chymotrypsin,2 to affinity gels consisting of 4-phenylbutylamine or ε-aminocaproyl-d-tryptophanmethylester covalently attached to Sepharose 4B was investigated. Considerable heterogeneity in the binding was observed, both at the level of the affinity adsorbent and with respect to the proteins themselves. Both the aromatic moiety of the ligand and the positively charged isourea group introduced during the coupling of the ligand to the gel play a part in the stabilization of the binding of α-chymotrypsin. At least a part of the binding involves the substrate binding site of the enzyme. The existence of secondary binding sites on the proteins, capable of interaction with gel-bound ligand, is suggested by the observation that at fairly high levels of substitution of gel by ligand the zymogen and covalently modified enzyme species are also bound quite strongly. Since the level of gel substitution is difficult to control, the results emphasize the necessity of a thorough investigation of the binding properties of a given affinity medium prior to use in the separation of active chymotrypsin from inactive derivatives.

Keywords

Chymotrypsin Column Volume Substrate Binding Site Cyanogen Bromide Affinity Adsorbent 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

ACTME

ε-aminocaproyl-d-tryptophnemethylester

PBA

4-phenyl-butylamine

DFP

diisoproylfluorophosphate

TPCK

l-(l-tosylamido-2-phenyl) ethylchloromethyl ketone

ATEE

N-acetyltyrosine ethyl ester

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Copyright information

© Humana Press Inc. 1978

Authors and Affiliations

  • George Tomlinson
    • 1
  • W. Charles Schellenberg
    • 1
  • T. Viswanatha
    • 2
  1. 1.Department of ChemistryUniversity of WinnipegWinnipeg
  2. 2.Department of ChemistryUniversity of WaterlooWaterloo

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