A method for obtaining linear reciprocal plots with caeruloplasmin and its application in a study of the kinetic parameters of caeruloplasmin substrates

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Abstract
1. The curved plots of 1/v against 1/[S] obtained when caeruloplasmin oxidizes NN-dimethyl-p-phenylenediamine were investigated. The first free-radical oxidation product of caeruloplasmin oxidation of NN-dimethyl-p-phenylenediamine is required for curvature, as straight-line plots were obtained when activities were measured either before appreciable free-radical product had appeared or in the presence of ascorbate, which reduced it back to NN-dimethyl-p-phenylenediamine. 2. In the presence of ascorbate linear reciprocal-plots were obtained with all of the 37 substrates tested. Vmax. values varied over only an eightfold range and those for the 20 p-amino compounds over only a twofold range. Km values, however, varied over a 104-fold range. The small range of Vmax. values indicates that the rate-limiting step in caeruloplasmin action is relatively independent of the nature of the substrate. Km values suggest that substrates bind primarily by ring electrons, although certain side-chain groups increased the Km in a manner unrelated to likely changes of ring-electron densities. A mechanism involving repulsion between negative charges on the substrate and the enzyme was supported by the variation of the Km of 5-hydroxyindol-3-ylacetic acid with pH.