Purification and fluorometric assay of proteinase A from yeast.
Proteinase A was purified from commercial baker's yeast to homogeneity by using affinity chromatography. Simple and sensitive fluorometric assay procedures were developed for this enzyme, where Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide and dimethylcasein were used as synthetic substrate and protein substrate, respectively. Proteinase A cleaved the former substrate at the Leu-Val bond. The extent of the cleavage was monitored by measuring the amount of fluorescent 7-amino-4-methylcoumarine produced by the successive cleavage with an auxiliary enzyme, aminopeptidase M, or by measuring the fluorescence generated by the reaction of newly formed amino groups with fluorescamine. In the assay with the latter substrate, the amounts of newly formed amino groups were measured by the reaction with fluorescamine. The optimal pH of the reaction was 5.0 to 5.5. The methods were applied to the study of the effects of denaturing agents on the activity of proteinase A.
D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence
D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases