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Use of Protein Difference Spectrophotometry to Determine Enzyme-Cofactor Dissociation Constants

1963; Elsevier BV; Volume: 238; Issue: 12 Linguagem: Inglês

10.1016/s0021-9258(18)51836-6

1083-351X

Clarence H. Suelter, Wayne R. Melander,

Metabolism and Genetic Disorders

The technique of difference spectrophotometry, first applied to proteins by Laskowski et al. ( 1) and further exploited by several groups of workers, as reviewed recently by Wetlaufer (2) and Reaven (3), has been applied primarily to problems in which changes in protein conformat'ion are involved.These studies are based on observations made with the individual amino acids, phenylalanine, tyrosine, and tryptophan.From the effects of solvents, urea, salts, and pH on the difference spectra of these chromophores, conclusions can be made concerning t)he environment of these chromophores in a protein molecule.This report constitutes the first case to our knowledge in which a protein difference spectrum due to the perturbation of one of the chromophores of the protein molecule caused by the specific interaction of a cofactor was used to determine the dissociation constant for that cofactor.This difference spectrum is to be contrasted with the changes in the spectra of coenzymes such as FAD, NAD+, or NADH that are often observed when these are bound to an enzyme.In this case, when varying amounts of the cofactor, Mn++ or Mg++, were added to the enzyme, increasing differences in the spectrum of the enzyme were observed until the enzyme became saturated.The plot of the change in absorbance against the negative log of the concentration of the cofactor permits an estimation of the dissociation constant (4).Pyrurat,e kinase (EC 2.7.1.40), the enzyme used for this study, was prepared by the method of Tietz and Ochoa (5).The specific activity of each preparation was determined by measuring the pyruvic acid formed with the dinitrophenylhydrazine method of Friedemann and Haugen (6) as adapted to pyruvate kinase by Reynard et al. ( 7).These activities were variable, ranging from 130 to 300 pmoles of pyruvate produced per minute per mg of enzyme at 25".Fig. 1 shows a series of difference spectra that are obtained at varying levels of manganese (II) chloride.These spectra were recorded on the same protein solution after t'he addition of microliter quantities of 0.01 M and 0.1 M h1nC12 to the sample cuvette and the same volume of either 0.03 M or 0.3 M tetramethylammonium chloride to the reference cuvett.e to maintain the same ionic strengt,h.Each spectrum was determined wit,h a Cary model 15 recording spectrophotometer and represents an average of four to five tracings with the recorder at 0.0 to 0.1 absorbance full scale.