Trypsin enzymatic hydrolysis

Partial hydrolysis of a peptide can be carried out either chemically with aqueous acid or enzymatically. Acidic hydrolysis is unselective and leads to a more or less random mixture of small fragments, but enzymatic hydrolysis is quite specific. The enzyme trypsin, for instance, catalyzes hydrolysis of peptides only at the carboxyl side of the basic amino acids arginine and lysine chymotrypsin cleaves only at the carboxyl side of the aryl-substituted amino acids phenylalanine, tyrosine, and tryptophan. 

There is currently little understanding of the influence of interfacial composition and (nano)structure on the kinetics of enzymatic hydrolysis of biopolymers and lipids. However, a few preliminary studies are beginning to emerge (McClements et al., 2008 Dickinson, 2008). Thus, for example, Jourdain et al. (2009) have shown recently that, in a mixed5 sodium caseinate + dextran sulfate system, the measured interfacial viscosity increased from qs = 220 mN s m 1 without enzyme to qs = 950 mN s m 1 with trypsin present. At the same time, the interfacial elasticity was initially slightly reduced from (7S = 1.6 mN m 1 to (h = 0.7 mN m, although it later returned to close to its original value. Conversely, in the... 

Selective enzymatic hydrolysis. The traditional strategy in sequence determination is to cut protein chains into smaller pieces which can be separated by chromatography or electrophoresis and sequenced individually. Enzymatic cleavage is especially useful because of its specificity. Trypsin, a so-called endo-... 

Total thiamine Milk Enzymatic hydrolysis of protein with trypsin and thiamine phosphates to thiamine with claradiastase oxidation of thiamine to thiochrome using ferricyanide (derivatization stopped with sodium sulphite) thiochrome extracted with 1-butanol Analytical Nucleosil Phenyl (150 mm, 5 fi Macherey-Nagel). Isocratic methanol + acetonitrile + isobutanol + water (80 +10+10+5 v/v/v/v). 0.7 ml/min. Fluorescence 375/430 nm (ex/em). External standardization. 76 Recoveries 95% thiamine as thiochrome from milk. 

Dissolution and Separation by Special Applications Enzymes and Microextraction Several enzymes such as trypsin, protease type XIV, lipase and/or cellulase, are used for enzymatic hydrolysis. For the determination of Hg2+ and Me-Hg in fish... 

Enzymatic hydrolysis Increased the soluble protein, compared to the respective controls (Figure 13). Approximate threefold Increases were noted after pepsin digestion, fourfold after bromelain, and twofold after trypsin. These levels of protein solubility were evident after only 10 min of enzyme treatment. Further digestion times did not significantly Increase the amount of soluble protein. 

Figure 1. Difference spectra for soy 7S protein modified by enzymatic hydrolysis (----------------) by trypsin at pH 7.1 and (---) by rennin at pH 5.8...

One of the earliest suggestions that total enzymatic hydrolysis was possible came from the studies of Frankel (1916), who showed that over 90 % of the bonds in several proteins could be broken when proteolysis with pepsin, trypsin, and chymotrypsin was followed by prolonged hydrolysis with the erepsin preparation of Cohnheim (1901). The recognition in later years of several peptidases in intestinal exti acts which will specifically act upon bonds that are not susceptible to the endopoptidases (Bcrg-mann, 1942) probably accounts for these obseiwations. The specific peptidases such as prolidase, iminodipeptidase (prolinase), glycylglycine dipeptidase, tripeptidase, and leucine aminopeptidase, whi( h are present in mucosa, attack many of the bonds that resist the action of endopoptidases. 

One method of sample preparation proposed for MS/MS analysis of proteins is by performing enzymatic hydrolysis of gel pieces from SDS-PAGE (see paragraph 7.3.1) using trypsin, proteins are hydrolyzed at the carboxyl side of lysine and arginine residues. Protocol of enzymatic digestion and sample preparation is reported in Table 7.8. 

Enzymatic hydrolysis (trypsine) carried out for fauna samples sediments were treated with 1 mol L H3PO4 in an open focused MW system separation on anion-exchange column (Hamilton PRP-XlOO) step gradient elution with a phosphate-based mobile phase, pH 6-7.5 on-line ICP-MS... 

We have shown by a comparison of the pH dependence of the step characterized by ki that the hydrolysis of the enzyme-acyl compound is the rate-determining step for the enzymatic hydrolysis of the usual amino acid amide substrates. In the case of chymotrypsin, acetyl-L-phenylalanine ethyl ester is hydrolyzed 1,000 times faster than the corresponding amide and in the case of trypsin, benzoyl-L-arginine ethyl ester is hydrolyzed 300 times faster than the corresponding amide. This suggests that for the amide hydrolysis too the second step, the acylation of the enzyme, must be the rate-determining step, since the third step is obviously identical for esters and amides of the same amino acid derivatives. The pH dependence of the chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester and acetyl-L-phenylalanine ethyl ester indicates that for these reactions ki and kz are of the same order of magnitude and both contribute to the over-all rate, as shown by Equation (4). 

The enzymatic hydrolysis of n-benzoyl L-arginine ethyl ester (BAEE) was carried out in a packed bed using trypsin bound to particles of porous glass. The glass was 200 00 mesh with 355-A pore diameter, and the bed had a 0.9-cm diameter and was 2.2 cm high (see Table 1.13). 

For this reason, glycoproteins must first be isolated from the biological matrix by dialysis, preparative chromatography, isoelectric focusing, and so forth or by a combination of several methods. For a structural determination, degradation steps such as a site-specific proteolysis (e.g., with trypsin), removal of oligosaccharides from the polypeptide (by an enzymatic hydrolysis or hydrazine... 

Claradiastase and trypsin in phosphate buffer have been used for hydrolysis of sample matrix for the determination of supplemental folic acid (27). Jacoby and Henry (26) further modified the method of Hoppner and Lampi (41) for folic acid by HPLC, in which the folic acid added to infant formulas and liquid medical nutritionals is quantitatively extracted with the aid of bacterial protease and papain. One disadvantage of the enzymatic extraction method was the large number of UV-absorbing compounds that are formed during enzymatic hydrolysis. These can interfere in the quantitation of the folate peaks. Thus, addition of a-amylase and protease to enhance extraction has not been common practice for methods involving HPLC. However, Pfeiffer et al. (13) suceessfiilly used triple-enzyme treatment prior to HPLC analysis with a purification method based on affinity chromatography. 

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