Comparison of the Hide Powder Azure and Casein-Trinitrobenzene Sulfonic Acid Methods for Determining Proteolysis in Skim Milk

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Abstract
Three extracellular psychrotroph proteinases were assayed in a defined buffer system and in skim milk, using the insoluble protein-dye complex, hide powder azure (HPA), as well as by quantifying free amino groups released from casein with trinitrobenzene-sulfonic acid (TNBS). Apparent Km values for HPA of approximately 60 mg/ml were found for each enzyme while for sodium caseinate, the apparent Km of 9.14 mg/ml for enzyme ATCC 15456 was 3-fold greater than for enzymes 13 and 32A. Vmax values for enzyme ATCC 15456 were 3 to 3.5 and 1.6 to 2.8-fold lower than for the other two enzymes with HPA and sodium caseinate, respectively. When activities in skim milk were being determined, trichloroacetic acid (TCA) concentrations of 2 and 8% for HPA and TNBS, respectively, were chosen to maximize recovery of degradation products and minimize background. Proteolysis measurements in skim milk with the two methods were closely correlated (r = 0.903 to 0.920) for each of the three enzymes. Detectability values (obtained...