An inexpensive device for sample stirring in VSU Zeiss spectrophotometers. Its use for kinetic measurements and active site titrations with immobilized enzymes.
The construction of a simple and effective sample stirring device for commercial spectrophotometers and its use for continuous kinetic measurements and active site titrations with immobilized enzymes is described. Sepharose-bound leucine aminopeptidase and trypsin were selected as model enzymes to test the performance of the magnetic stirring equipment. Kinetic parameters of insolubilized leucine aminopeptidase using L-leucine p-nitroanilide as substrate and the catalytic site concentration of matris-bound trypsin using p-nitrophenyl p'-guanidinobenzoate as active site titrant could be evaluated without significant interference from the turbidity of the stirred Sepharose suspension. The problem of grinding of the support material could be overcome. Both unbound native and carrier-fixed enzyme may be reacted under identical conditions with similar convenience and sensitivity.
D000818:Animals; D001665:Binding Sites; D002021:Buffers; D002417:Cattle; D007202:Indicators and Reagents; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology; D007931:Leucyl Aminopeptidase / Q000378:metabolism*; D012685:Sepharose; D012995:Solubility; D013053:Spectrophotometry / Q000295:instrumentation*; D014357:Trypsin / Q000378:metabolism*
D002021:Buffers; D007202:Indicators and Reagents; D012685:Sepharose; D007931:Leucyl Aminopeptidase; D014357:Trypsin