A Rationale for Stabilization of Oxygen-Labile Enzymes: Application to a Clostridial Hydrogenase

A general procedure for stablization of O2-labile enzymes exploiting ``salting out'' of oxygen from the microenvironment in the molecular layers immediately adjacent to charged surfaces of polyionic solid adsorbents has been developed. Empirical verification of this rationale is provided....

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1978-08, Vol.75 (8), p.3640-3643
Main Authors: Klibanov, Alexander M., Kaplan, Nathan O., Kamen, Martin D.
Format: Article
Language:English
Subjects:
Adsorbents
Adsorption
Anions
Biochemistry
Clostridium - enzymology
Enzymes
Enzymes, Immobilized
Half lives
Oxidoreductases
Oxygen
Salts
Solutions
Solvents
Viologens
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Summary:A general procedure for stablization of O2-labile enzymes exploiting ``salting out'' of oxygen from the microenvironment in the molecular layers immediately adjacent to charged surfaces of polyionic solid adsorbents has been developed. Empirical verification of this rationale is provided. The half-life of air inactivation of the O2-labile hydrogenase (EC 1.12.7.1) from Clostridium pasteurianum is increased 20- to 25-fold simply by adsorption (noncovalent binding) in dilute Tris· HCl buffer on common anion exchange supports such as DEAE-cellulose or Dowex 1-X2. Predicted increases in degree of stabilization by using more densely charged adsorbents (such as polyethyleneimine-cellulose), as well as bulkier solvent counter-anions, are found; half-lives for air inactivation for the bound hydrogenase can be increased to 3000-fold longer than that of the free enzyme. Most of the total catalytic activity, assayed as H2evolution from dithionite mediated by methyl viologen or ferredoxin, is retained, whereas the expected suppression of H2uptake in the reverse reaction is observed.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.75.8.3640