Bromelain assay

Application and Principle This procedure is used to determine the proteolytic activity of papain, ficin, and bromelain. The assay is based on a 60-min proteolytic hydrolysis of a casein substrate at pH 6.0 and 40°. Unhydrolyzed substrate is precipitated with trichloroacetic acid and removed by filtration solubilized casein is then measured spectrophotometri-cally. 

The most used synthetic substrates are V-benzoyl-L-Arg-ethyl ester (BAEE), N-benzoyl-L-Arg-amide (BAA), N-benzyloxycarbonyl-Lys-p-nitrophenyl ester, N-benzoyl-L-Arg-p-nitroanilide (L-Bapa), tosyl-L-Arg-methyl ester (TAME), iV-benzoyl-L-Arg-methyl ester (BAME), Gly-ethyl ester, His-ethyl ester, benzoyl-Gly-ethyl ester, L-Leu-p-nitroanilide (Leupa), L-Ala-p-nitroanilide (Alapa), L-Phe-p-nitroanilide (Phenpa), W-benzyloxycarbonyl-Arg-Arg-p-nitroanilide and jV-benzyloxycarbonyl-Arg-Arg-7-(4-methyl)coumarinylamide. Substituted ben-zoylglycine-methyl ester (substituted in position 3 or 4 of benzene, e.g., 3-cyano hippuric acid methyl ester, and N-succinyl-Ala-Met-S-benzyl-Cys-p-nitroanilide are also sensitive substrates for assaying bromelain [14,19,24,33,35,38-41] (Table 1). 

For historical reasons many pharmaceutical enzymes are assayed with physiological or biopolymeric substrates (proteins, polysaccharides, bacteria, oil emulsions), which causes a number of theoretical and practical problems. The interpretation of results is difficult when natural substrates are converted into products that are substrates themselves for the next enzymatic attack. Reaction rates often depend on the position of the scissile bonds in the molecule and the chemical nature of the moieties. Hydrolysis can proceed simultaneously on various bonds at various rates. In proteolysis it is assumed that some products are liberated only after denaturation and that during the reaction course new peptide bonds become accessible for hydrolysis. In these cases the enzymatic mechanisms become exceedingly complex, kinetic parameters are apparent values, and experimental results are strongly influenced by the reaction conditions. Reproducibility problems can occur upon assaying proteinases with a limited specificity for particular casein types. Bromelain and pancreatic proteinase, FEP pharmaceutical enzyme standards, are assayed with a casein substrate. The extent of soluble peptide release is a measure of proteolytic activity. However, due to limited specificity, some proteinases release peptides with a nonrandom aromatic amino acid composition. Contamination of casein preparations with protein and of test enzyme substances with other proteinases biases the assay results. Under these conditions, relative assay methods are indicated. 

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