Abstract
A general procedure for stabilization 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 H2 evolution from dithionite mediated by methyl viologen or ferredoxin, is retained, whereas the expected suppression of H2 uptake in the reverse reaction is observed.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Abeles F. B. Cell-free Hydrogenase from Chlamydomonas. Plant Physiol. 1964 Mar;39(2):169–176. doi: 10.1104/pp.39.2.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Benemann J. R., Berenson J. A., Kaplan N. O., Kamen M. D. Hydrogen evolution by a chloroplast-ferredoxin-hydrogenase system. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2317–2320. doi: 10.1073/pnas.70.8.2317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berenson J. A., Benemann J. R. Immobilization of hydrogenase and ferredoxins on glass beads. FEBS Lett. 1977 Apr 1;76(1):105–107. doi: 10.1016/0014-5793(77)80130-0. [DOI] [PubMed] [Google Scholar]
- Chen J. S., Mortenson L. E. Purification and properties of hydrogenase from Clostridium pasteurianum W5. Biochim Biophys Acta. 1974 Dec 18;371(2):283–298. doi: 10.1016/0005-2795(74)90025-7. [DOI] [PubMed] [Google Scholar]
- GOLDSTEIN L., LEVIN Y., KATCHALSKI E. A WATER-INSOLUBLE POLYANIONIC DERIVATIVE OF TRYPSIN. II. EFFECT OF THE POLYELECTROLYTE CARRIER ON THE KINETIC BEHAVIOR OF THE BOUND TRYPSIN. Biochemistry. 1964 Dec;3:1913–1919. doi: 10.1021/bi00900a022. [DOI] [PubMed] [Google Scholar]
- Jones L. W., Bishop N. I. Simultaneous measurement of oxygen and hydrogen exchange from the blue-green alga anabaena. Plant Physiol. 1976 Apr;57(4):659–665. doi: 10.1104/pp.57.4.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karube I., Matsunaga T., Tsuru S., Suzuki S. Continuous hydrogen production by immobilized whole cells of Clostridium butyricum. Biochim Biophys Acta. 1976 Sep 24;444(2):338–343. doi: 10.1016/0304-4165(76)90376-7. [DOI] [PubMed] [Google Scholar]
- Lappi D. A., Stolzenbach F. E., Kaplan N. O., Kamen M. D. Immobilization of hydrogenase on glass beads. Biochem Biophys Res Commun. 1976 Apr 19;69(4):878–884. doi: 10.1016/0006-291x(76)90455-1. [DOI] [PubMed] [Google Scholar]
- Nakos G., Mortenson L. Purification and properties of hydrogenase, an iron sulfur protein, from Clostridium pasteurianum W5. Biochim Biophys Acta. 1971 Mar 10;227(3):576–583. doi: 10.1016/0005-2744(71)90008-8. [DOI] [PubMed] [Google Scholar]
- Packer L. Problems in this stabilization of the in vitro photochemical activity of chloroplasts used for H2 PRODUCTION. FEBS Lett. 1976 Apr 15;64(1):17–19. doi: 10.1016/0014-5793(76)80237-2. [DOI] [PubMed] [Google Scholar]
- Rao K. K., Rosa L., Hall D. O. Prolonged production of hydrogen gas by a chloroplast biocatalytic system. Biochem Biophys Res Commun. 1976 Jan 12;68(1):21–28. doi: 10.1016/0006-291x(76)90004-8. [DOI] [PubMed] [Google Scholar]