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Characterization of heterogeneous immobilized enzyme subpopulations using EPR spectroscopy
Author(s) -
Clark Douglas S.,
Bailey James E.
Publication year - 1984
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260260306
Subject(s) - electron paramagnetic resonance , chemistry , enzyme , immobilized enzyme , sepharose , molecule , enzyme assay , spectral line , chymotrypsin , indole test , solvent , chromatography , stereochemistry , organic chemistry , nuclear magnetic resonance , trypsin , physics , astronomy
Direct evidence was obtained for the existence of two distinct forms of active α‐chymotrypsin immobilized on CNBr‐activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectra of five different spin‐labeled immobilized enzyme formulations in the presence of indole were all resolved into the same two spectral components. Both subpopulation spectra were approximately identified experimentally, and the subpopulation exhibiting greatly restricted spin‐label motion was shown also to be relatively inaccessible to solvent. Using overall specific activity data and subpopulation fractions from EPR spectral analysis, the specific activity of the more constricted immobilized enzyme active form was shown to be approximately 15 times smaller than that of the other class of immobilized enzyme molecules with an indole EPR spectrum similar to that of chymotrypsin in solution. Variations in overall specific activity of formulations with different loadings and different supports results entirely from changes in the proportions of the same two subpopulations of immobilized enzyme molecules.