A simple device for automated spectrophotometric kinetics using a diode array spectrophotometer.
In this report we describe an automated system that rapidly and automatically mixes reagents and records results, such as spectrophotometric changes. It employs a commercial diode array spectrophotometer and a novel dilution chamber in a flow stream that allows repetitive spectrophotometric rate measurements at accurately measured incremental substrate concentrations. When applied to enzyme kinetic studies, initial velocities at 15 different substrate or inhibitor concentrations, or pH values, can be recorded in a few minutes with high reproducibility, i.e., standard deviations less than 1%, and high sensitivity. Reactions occur in an 8-microliters flow cell and the reagent consumption is minimal. The concentration of incrementally diluted reagent in the cell is measured directly by means of an indicator dye added to the substrate. Michaelis-Menten parameters, inhibition constants, and pH profiles are determined for several enzymes including dehydrogenases producing NADH, a kinase requiring a coupled assay, and a hydrolase, carboxypeptidase A, in a reaction that produces a small decrease in absorbance.
Scopes
R K
RK
Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Holmquist
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D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D001322:Autoanalysis; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000032:analysis*; D010084:Oxidation-Reduction; D011770:Pyruvate Kinase / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation / Q000379:methods*; D014737:Vibrionaceae / Q000201:enzymology
D007527:Isoenzymes; D009243:NAD; D000426:Alcohol Dehydrogenase; D011770:Pyruvate Kinase