Trypsin inhibitor, solution preparation

Protein molecular weight standards—Suggested standards are phosphorylase (97,400 Da), bovine serum albumin (66,200 Da), ovalbumin (45,000 Da), carbonic anhydrase (31,000 Da), soybean trypsin inhibitor (21,500 Da) and lysozyme (14,400 Da) at 1 mg/ml each in a single mixture in 0.01 M Tris chloride buffer, pH 7.0 (prepared by dilution of the stock 1 M buffer above). Approximately 2 ml of this solution will be required. 

Trypsin preparations are often contaminated by chymotrypsin, which can be inactivated by treatment with L-(l-tosylamino-2-phenyl)ethylchloromethyl ketone (TPCK). Trypsin in solution should be stored in 10 mM HC1, otherwise it will undergo autoproteolysis. For proteolytic cleavage, trypsin is dissolved (freshly) in 100 mM Na bicarbonate buffer and added in a proportion of 2-10% (w/w) of the protein substrate. Reaction can be stopped by addition of phenylmethylsulphonyl-fluoride (PMSF) or soybean trypsin inhibitor (4 mg inhibitor per mg trypsin). 

Russet Burbank potatoes were quartered and placed in a water bath at 80 C for 10 min. The tubers were rinsed with cold water and blended to a paste. Absolute ethanol was blended in slowly with the paste until it was 80% ethanol by volume. The mixture was filtered through Whatman No. 1 filter paper with the aid of a vacuum. The ethanol in the filtrate was removed by vacuum evaporation and the resulting solution was dialyzed in a 2,000 MW cut off dialysis bag against several changes of distilled water. The remaining extract in the dialysis bag was lyophilized. The dry material contained one major polypeptide, CPI, and some minor components. On a protein basis the material was over 95% CPI. Purity of this crude CPI fraction was also demonstrated by electrophoresis by the method of Panyim and Chalkley (8). No Inhibitor I nor Inhibitor II could be detected by immunological techniques, however a small quantity of trypsin inhibitor activity (less than 0.5%) was present, presumably from contamination by the polypeptide trypsin inhibitor that is present in potato tubers (4). This CPI preparation was utilized for the chick feeding experiments. 

The effect of EDTA on the rate of depolymerization and esterase activity was measured by the use of EDTA solutions instead of cholate buffer. The EDTA solution was neutralized to pH 7.0 and the final EDTA concentrations used were 0.025 M, 0.05 M, 0.075 M, 0.1 M, 0.15 M, 0.175 M and 0.2 M. EDTA controls without enzyme were also prepared. The effect of soybean trypsin inhibitor (SBTI) was also studied by using 0.25 mg/ml 0.5, 1, 1.5, 2.0 and 2.5 mg/ml of the inhibitor in the reaction mixture. 

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