A micromethod for determination of proteolytle enzymes in the pH range of 2.8 to 4.8: Radial enzyme diffusion into skim milk-containing agarose gel
✍ Scribed by H. Løwenstein; Agnete Ingild
- Publisher
- Elsevier Science
- Year
- 1976
- Tongue
- English
- Weight
- 608 KB
- Volume
- 71
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
Radial diffusion of enzymes into a casein-containing gel is a fast and inexpensive technique for determination of proteolytic enzymes. A simple method for the preparation of substrate plates with a homogeneous distribution of caseinate and buffered at selected pH values between 2.8 and 4.8 has been described in this communication. By determination of pepsin in caseinate gels the accuracy was better than that described for photometric pepsin assays, and the sensitivity was 300 times greater.
A sensitive and highly reproducible method for determination of milkclotting enzymes above pH 5.2 has been described by Lawrence and Sanderson (1). The method is based on radial diffusion of enzymes into a thin layer of agarose gel containing purified calcium caseinate. During investigations into the activity and the pH-profile of proteolytic enzymes, we found that the method of Lawrence and Sanderson could be modified to cover also the pH range of 2.8 to 4.8. Furthermore, the method could be simplified by using ordinary skim milk instead of purified calcium caseinate in the gel. In the present paper, porcine pepsin has been used for the demonstration of the technique as it is representative for a group of enzymes where time consuming photometric methods (2-4) can advantageously be substituted by the simple and inexpensive radial diffusion method.
Methods
Pepsin. Porcine pepsin, twice crystallized, was obtained from Worthington Biochemical Corporation, Freehold, N.Y. Agarose gel. One gram of agarose (Litex, Glostrup, Denmark) was dissolved in 100 ml of boiling water. The gel was kept above 55°C in a thermostated waterbath.
Skim milk. One gram of skim milk powder was reconstituted by gradually adding 30 ml of sodium acetate buffer, pH 5.5, ionic strength 0.10. 204