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Several methods for determining the activity of Proteinase K

2020-07-24 来源:亚科官网

Proteinase K (CAS:39450-01-6) has a stable structure, high enzyme activity and wide substrate specificity, and is widely used in related experiments in the fields of molecular biology and cell biology, as well as in industries and agriculture. This article will introduce several methods that can be used to detect proteinase K activity.

 Folin-Phenol method

This method is a common method for determining the activity of protease. The principle is that the Folin-phenol reagent can be reduced by phenolic compounds to give a blue color (molybdenum blue and tungsten blue mixture) under alkaline conditions, because the protein molecule contains sweat phenolic amino acids (Such as tyrosine, tryptophan, etc.), can make the protein and its hydrolysate undergo the above reaction. Therefore, this principle can be used to determine protease activity.

Under certain temperature and pH conditions, the proteinase K uses casein or denatured hemoglobin as the substrate. After a period of time, the enzymatic reaction is terminated. After centrifugation or filtration to remove the complex protein and other precipitates, the supernatant is taken and alkalized Afterwards, Folin-phenolreagent was added to develop the color. The depth of the blue color is proportional to the amount of tyrosine produced in the filtrate. Measure at 680 nm the amount of proteinase K (μmol) that is equivalent to the Folin-positive amino acid or peptide produced by tyrosine per minute.

UV spectrophotometry

Ultraviolet spectrophotometry is based on casein as a substrate, in a 0.01 mol/L Tris-HCl buffer at 55 ℃, pH 8.0, at a wavelength of 275 nm to determine the L-tyrosine produced by proteinase K hydrolyzing casein within 1 min The amount (μg).

Coomassie brilliant blue method

The method to determine the protein content is a kind of dye binding method. It is red in the free state, and the maximum light absorption is at 488nm; when it is combined with protein, it becomes cyan, and the protein-pigment conjugate has the maximum light absorption at 595nm wavelength. Its light absorption value is proportional to protein content, so it can be used for quantitative determination of protein. This method uses casein or bovine serum albumin as a substrate. Under certain temperature and pH conditions, proteinase K hydrolyzes casein or bovine serum albumin, and the dye Coomassie Brilliant Blue G-250 forms a blue complex with unhydrolyzed protein. The compound can be quickly measured at 595 nm to obtain proteinase K enzyme activity.

The above three methods are commonly used methods for determining the activity of proteinase K. They are useful for judging and comparing the activity of proteinase K provided by different manufacturers, so that people can choose and use proteinase K.

Related links: Proteinase K