Denaturation soluble enzyme assays

Table 2. Protocols used in attempts to correctly refold KAS I. Inclusion bodies were isolated fix)m induced E. coli cells containing expression constructs. Pellets were repeatedly washed in buffer containing 0.2% (w/v) Triton X-100 then in H2O. Protein was denatured in guanidine hydrochloride (2 M) except for the method of [9] in which the washed inclusion bodies were used directly. Results were assessed by enzyme assays or the appearance of soluble protein after removal of denaturant...

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. 

The activity of both proenzyme and active acid protease were assayed with acid-denatured hemoglobin substrate by the method of Kassell and Meitner (7) the proenzyme is converted to the active form in the acid conditions of the assay. The 2 ml assay mixture contained 0.1 M citrate-phosphate buffer, pH 2.5, 25 mg hemoglobin and appropriately diluted enzyme concentration. The assays were performed for 30 min at 37° and terminated by addition of 2 ml of 5% trichloroacetic acid the absorbance at 280 nm of soluble fraction was then measured. To assay proenzyme, the pH of the incubation was raised to 8 with 1 M Tris to destroy the active form. This solution was then acidified to activate and assay for the proenzyme. 

Nuclease activity is assayed by measuring the release of acid-soluble products from heat-denatured DNA (e.g., calf thymus). A typical assay mixture (0.5 ml) contains 30 mM NaAc (pH 4.6), 50 mM NaCl, 1 mM zinc acetate, 5% (v/v) glycerol, 0.5 mg/ml heat-denatured DNA, and enzyme. The assay mixture is incubated for 10 min at 37°C. 

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