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Use of Affinity Chromatography for Determination of Dissociation Constants of Complexes of Trypsin and Chymotrypsin with their Free and Immobilized Inhibitors

  • Jaroslava Turkova

Abstract

Since affinity chromatography is based in principle on the formation of a specific complex, e.g. between an enzyme and its competitive inhibitor, this method has become a tool of choice also for studies on specific interactions. For example, an enzyme can be adsorbed on a column of the immobilized inhibitor and eluted by solutions of the free inhibitor of different concentrations. If the elution volumes of the enzyme are plotted versus the inhibitor concentrations used for elution, then the dissociation constants of the complex formed by the enzyme and the soluble inhibitor (KI) and the enzyme and the immobilized inhibitor (KL) can be determined. It is necessary that the same inhibitor be used both immobilized on a carrier and soluble as eluant; then the effect of the carrier and the attachment on the specific interaction between the enzyme and the immobilized inhibitor can be estimated from comparison of the KI and KL values. The determination of both constants can be done using zonal analysis (1) or frontal analysis (2) type experiments. Making use of our previous experience with the isolation of chymotrypsin and trypsin on specific adsorbents prepared by attachment of natural high molecular weight inhibitors and low molecular weight synthetic inhibitors to Spheron (3-5), we decided to study the effect of carrier and attachment on the specific interaction of the enzyme with its immobilized inhibitor by these two methods.

Keywords

Dissociation Constant Affinity Chromatography Specific Interaction Elution Volume Specific Adsorbent 
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Copyright information

© Plenum Press, New York 1978

Authors and Affiliations

  • Jaroslava Turkova
    • 1
  1. 1.Institute of Organic Chemistry and BiochemistryCzechoslovak Academy of Science PragueCzechoslovakia

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