A rapid method for measuring protease activity in milk using radiolabeled casein.
A rapid means to detect the presence of protease activity in raw milk could be useful in predicting keeping ability of products made from that milk. A 30-min assay has been developed and compared with three other methods of detecting protease. Casein, [methyl-14C]-methylated-alpha was purchased from a radioisotope supplier. Concentrations of substrate from 2 to 20 nCi gave counts per minute, which increased linearly when counted with the Charm analyzer. There was not a significant difference in counting times of 10, 20, or 30 min. A mixture of sodium acetate and acetic acid precipitated nonhydrolyzed substrate with an efficiency of 97%. Comparison of the [14C] casein assay, a casein fluorescein isothiocyanate assay, trinitrobenzenesulfonic acid procedure, and the Hull procedure using protease from psychrotrophic bacteria revealed that the [14C] casein and casein fluorescein isothiocyanate methods were roughly equivalent and that the radiometric procedure was 10 times more sensitive than the trinitrobenzenesulfonic acid assay. The radiometric procedure was approximately 10(4) times more sensitive than the Hull procedure. The [14C] casein and casein fluorescein isothiocyanate methods were similar in time required, about 30 min, while the trinitrobenzenesulfonic acid assay and Hull method required about 1 h plus reagent preparation time. The [14C] casein procedure was most expensive per test; the other three were cheaper and similar to each other in cost.
D000150:Acinetobacter / Q000201:enzymology; D000818:Animals; D002250:Carbon Radioisotopes; D002364:Caseins / Q000032:analysis*; D002417:Cattle; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D066298:In Vitro Techniques; D008892:Milk / Q000032:analysis / Q000201:enzymology* / Q000382:microbiology; D010447:Peptide Hydrolases / Q000032:analysis*; D011551:Pseudomonas fluorescens / Q000201:enzymology; D013861:Thiocyanates; D014302:Trinitrobenzenesulfonic Acid
D002250:Carbon Radioisotopes; D002364:Caseins; D005452:Fluoresceins; D013861:Thiocyanates; D014302:Trinitrobenzenesulfonic Acid; D010447:Peptide Hydrolases; D016650:Fluorescein-5-isothiocyanate