Pronase, specificity

Enzymatic gelation of partially heat-denatured whey proteins by trypsin, papain, pronase, pepsin, and a preparation of Streptomyces griseus has been studied (Sato et al., 1995). Only peptic hydrolysate did not form a gel. The strength of the gel depended on the enzyme used and increased with increasing DH. Hydrolysis of whey protein concentrate with a glutamic acid specific protease from Bacillus licheniformis at pH 8 and 8% protein concentration has been shown to produce plastein aggregates (Budtz and Nielsen, 1992). The viscosity of the solution increased dramatically during hydrolysis and reached a maximum at 6% DH. Incubation of sodium caseinate with pepsin or papain resulted in a 55-77% reduction in the apparent viscosity (Hooker et al., 1982). 

Such isopeptides also have been found naturally present in keratin (77,78,85) and in polymerized fibrin (79). Their quantitative determination requires an enzymic hydrolysis using pepsin, pronase, amino-peptidase, and prolidase as described by Cole et al. (135) followed by a chromatographic separation using an amino acid analyzer under very specific conditions (80). 

After the introduction of pronase E, other more or less nonsubstrate-specific proteolytic enzymes have been applied to assist Se speciation. Most of them were derived from DNA/RNA clean-up protocols. The new enzymes (subtilisin from Bacillus licheniformis, also named protease VIII, EC 3.4.21.14 proteinase K from Tritirachium album, EC 3.4.21.64 the crude Novo Nordisk product of Flavourzyme from Aspergillus oryzae) proved to be capable of extracting Se with varying yields and chromatographic recovery of Se species. It is important to highlight that the latter parameter also depends on the instrumentation available. In this regard, different recovery values for the same samples reported by independent research groups do not necessarily indicate successful or unsuccessful sample preparation. Similarly, extraction efficiency (defined as the ratio of extracted Se to total Se in the sample) cannot be used as such for comparison purposes because sample preparation may include some extra steps, for example, TCA precipitation or ultrafiltration, which may reduce this value even by 10-20 percent. 

The authors of the studies cited above found that the enzymes used during sample preparation could not completely hydrolyze sample proteins into amino acids. This is partly understandable as both proteinase K and subtilisin are known to show hydrolyzing preference for specific residues of proteins. Accordingly, they do not necessarily arrive at total hydrolysis. At the same time, neither pronase E nor protease XIV could always provide 100 percent extraction of Se from the samples (see Table 19.1). Therefore, alternative methods should be developed in the field of sample preparation to replace enzymatic methods. 

Grafts isolated by pepsin and trypsin digestion had stronger amide absorption bands than after pronase digestion. Pronase, because of its broad specificity, is capable of hydrolyzing the collagen trunks more extensively than the other two enzymes. Thus, the grafts isolated by... 

Relative NA specific activity can be quantified using a combination of the NC-10 MAb-based ELISA for quantifying the relative amount of native protein and comparing this to the enzyme activity for the same dilutions in a MUNANA-based enzyme assay. Either purified NA prepared by pronase treatment of virus or whole virus can be used in both assays. 

Analysis after digesting protein A) Site specific digestion 1. Treatment with pronase Yields glycoamino acids or glycopeptides with a very short polypeptide chain [253]... 

Studies by Henderson and associates present another view of adhesion. Their results show that stage-specific EndoH-resistant membrane-derived pronase glycopeptides can partially block EDTA-resistant adhesion [145]. The glycopeptides could be derived from... 

Treatment of the reduced adduct with pronase and subsequent reduction with Raney nickel led to the release and isolation of trans-/3-methylstrene 39. Raney nickel is known to cleave carbon-sulfur bonds specifically (777). Furthermore, the expected product from cleavage of the carbon-sulfur bond of the reduced adduct is 1-phenyl-1-propanol however, under the conditions of the Raney nickel reduction, 1-phenyl-1-propanol is dehydrated to trans-/3-methylstyrene (770). This result suggests that the amino acid residue is cysteine. The finding that the total number of cysteine residues detected with 5,5 -dithiobis(2-nitro-benzoic acid) decreases by one in the denatured, 1-phenylcyclopropylamine-... 

Hydrolysis of whey protein concentrate (WPC) with pepsin, pronase or prolase decreased emulsifying capacity specific foam volume was increased by very limited hydrolysis (56,57), but decreased by more extensive hydrolysis, while foam stability was greatly decreased by limited hydrolysis (52) (Fig.3). 

---