NON-ENZYMIC PRODUCTION OF SOLUBLE COLORED PRODUCTS, FROM INSOLUBLE PROTEIN-DYE SUBSTRATES

摘要

The object of this study was to increase the reliability and use-fulness of proteinase assays using dyed insoluble protein substrates by determining the cause of positive tests in the absence of proteolytic activity and by investigating methods for differentiating between enzymic and non-enzymic color production.nSoluble colored products were formed when solutions of serum albumin were added to hide powder dyed with Congo red. The color production was pH dependent and proportional to the amount of albumin in the reaction mixture. Formation of color did not show the usual time dependence characteristic of enzyme activity and was not inhibit¬ed by boiling the solutions. The absorption spectrum of the soluble products was similar to that of an albumin-Congo red complex. The net increase in soluble colored material caused by addition of albumin could be eliminated by increasing the amount of hide powder. Degra¬dation of the substrate by trypsin, however, was proportional to the enzyme concentration and the interfacial area of the substrate.nFalse indications of proteolytic activity with insoluble protein-dye complexes result if the test solution contains soluble proteins having a strong affinity for the non-covalently bound dye of the sub¬strate. Non-enzymic contributions to the color production in the assay can be measured by taking advantage of differences in the mechanisms of the two processes.