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Determination of enzyme kinetic parameters by continuous addition of substrate to a single reaction mixture and analysis by a tangent slope procedure 2. application of the method to soluble and immobilized enzymes



Determination of enzyme kinetic parameters by continuous addition of substrate to a single reaction mixture and analysis by a tangent slope procedure 2. application of the method to soluble and immobilized enzymes



Analytical Biochemistry 104(2): 370-385



A system is described for semiautomated evaluation of enzyme kinetic parameters, S0.5 [half-maximal rate], V [maximal reaction velocity], and Hill n [slope]. It consists of a Gilford spectrophotometer modified for continuous addition of substrate to a stirred enzyme assay mixture, and for recording of absorbance data coded on paper tape. The Hill parameters for a number of enzymes, obtained from these data by tangent-slope or curve-fitting procedures, are in good agreement with published or manually determined values. Experiments with rabbit muscle lactate dehydrogenase covalently linked to Sephadex G-50 or Sepharose 4B demonstrate the feasibility of this approach when applied to enzymes attached to solid supports. Instrumental errors due to spectrophotometer nonlinearity and drift, stirring, mixing, pumping, truncation in readout of absorbance, and bias in experimental parameters are minimal and do not significantly interfere with the method. The advantages and disadvantages of the approach are discussed, and improvements and extensions are suggested for the instrumental and data handling system which could greatly extend its utility.

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Accession: 005122874

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PMID: 7446963


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