Sep 1, 1976

Active enzyme gel chromatography. I. Experimental aspects

Biophysical Chemistry
M M JonesG K Ackers

Abstract

Transport properties of active enzyme species can be studied effectively by layering a small band of enzyme-containing sample on a gel chromatographic column previously saturated with substrate. The column is optically scanned at successive time intervals to yield profiles representing the appearance of chromophoric product or disappearnce of chromophoric substrate. These profiles permit determination of the specific activity and rate of transport of the active species. Initial studies on mechanic of the technique establish the feasibility of accurately determining transport properties of active enzyme species chromatographed on gel columns. Illustrative results are presented for L-glutamate dehydrogenase and for homoserine dehydrogenase studied in both forward and reverse reactions. It is shown that the partititon cross sections derived from the rates of motion of catalytic activity are the same as those determined by equilibrium saturation experiments which directly measure the degree of partitioning by the protein. These results establish the validity of the technique for a variety of future studies. Active enzyme gel chromatography appears comparable in precision to the active enzyme sedimentation technique at current stage...Continue Reading

  • References4
  • Citations5

Mentioned in this Paper

Anatomical Layer
Alcohol Oxidoreductases
Gel Chromatography
Enzyme Activity
Sedimentation Procedure
NADP
Molecular Sieve Chromatography
GLUD1
L-glutamate Dehydrogenase
Mathematics

About this Paper

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