Tryptic hydrolysis

The T2 site also became protected from tryptic hydrolysis after phosphorylation of the native or solubilized sarcoplasmic reticulum vesicles with inorganic phosphate in a calcium free medium in the presence of dimethylsulfoxide or glycerol [121,252]. Under these conditions the Ca -ATPase is converted into a covalent E2-P intermediate, that is analogous in conformation to the E2V intermediate formed in the presence of vanadate. In contrast to this, the T2 site in the stable phosphorylated Ca2E P intermediate generated by the reaction of the Ca -ATPase with chro-mium-ATP in the presence of Ca [178,253] was fully exposed to trypsin, just as it was in the nonphosphorylated Ca2Ei form. Therefore the phosphorylated intermediates show the same sensitivity to trypsin at the T2 site as the corresponding nonphosphorylated enzyme forms. 

DEAE 25W were operated in tandem to map the peptides generated on tryptic hydrolysis of very large proteins, such as albumin genetic variants.197 A useful review that covers many aspects of peptide analysis is available.198... 

Pathy, L., and Smith, E.L. (1975) Reversible modification of arginine residues Application to sequence studies by restriction of tryptic hydrolysis to lysine residues./. Biol. Chem. 250, 557. 

Glycine A-methyltransferase is also reported to have an ordered binding mechanism with SAM binding first to the enzyme, there being no metal-ion dependency. Cooperative behavior is observed with SAM binding. The cooperative nature can be eliminated by the tryptic hydrolysis of the N-terminal eight amino acid residues. 

Briand, L., Chobert, J.-M., and Haertle, T. 1994. Tryptic hydrolysis of esters of (3-casein and... 

Bouhallab, S., Molle, D., and Leonil, I. 1992. Tryptic hydrolysis of caseinomacropeptide in membrane reactor preparation of bioactive peptides. Biotechnol. Lett. 14, 805-810. 

Gagnaire, V., Pierre, A., Molle, D., and Leonil, J. 1996. Phosphopeptides interacting with colloidal calcium phosphate isolated by tryptic hydrolysis of bovine casein micelles. J. Dairy Res. 63, 405-422. 

Table 3. Kinetic parameters for tryptic hydrolysis of inverse and normal type substrates ...

Tryptic hydrolysis of cytochrome yields a polypeptide fragment which carries the heme and has a molecular weight of approximately... 

Mapping of the primary structure of CHO IL-4 was carried out by tryptic hydrolysis followed by measurement of the resulting peptide fragments by on-line HPLC/ESI-MS. Since the primary sequence of IL-4 was known, this MS mapping approach could confirm the cDNA-derived protein sequence,... 

The primary structure of the t chain is not established, and only results of preliminary structural analyses have been reported (H30, K15). Extensive studies of the chain have revealed rather unique structural features (C2). Its amino acid composition, for instance, differs from those of all other hemoglobin chains and is, among others, characterized by the presence of five isoleucyl residues. Tryptic hydrolysis produced several peptides with amino acid compositions not observed for any of the tryptic peptides of the a, y, or 8 chain, although other peptides had compositions identical to those of tryptic peptides of the y chain. There seems to be no doubt that the structures of the e and chains are different (C2). 

This positively-charged derivative is prepared from cysteine and ethylenimine ( 3.8.4) and is often used to introduce additional sites of tryptic hydrolysis into proteins. SAEC has a mobility of 2.07 compared to 1.00 for aspartic in pH 1.9 electrophoresis, and elutes from a 20 cm (basic) column of a Beckman analyzer at 82 ml compared to 74 ml for lysine and 96 ml for histidine (Schroeder et al. 1967). SAEC has a color... 

The combination of immobilized enzyme columns with HPLC/thermospray MS can be very useful in peptide identification and sequencing [6,7], There are a number of ways of combining the immobilized enzyme column, HPLC and MS detection for peptide analysis. Use of an endopeptidase column prior to HPI.C separation and MS detection will enable separation of each hydrolysis product for Identification. Figure 2 shows the trypsin column/HPLC/thermospray MS of Y-endorphin. The selected ion chromatograms show the retention time for each tryptic hydrolysis product Tj and T. Typically, this column configuration can only be used on purified samples since no separation or column clean-up is performed before hydrolysis. 

The use of thrombin, an enzyme that serves several roles in the blood-coagulating process, is a useful adjunct to tryptic hydrolysis. Its action is more specific and it cleaves only a limited number of arginyl bonds as a rule. Some arginyl bonds are only slowly hydrolysed by thrombin so that enzymic digests of protein can be quite complex in composition because degradation of substrate is incomplete. 

Panyam, D., and Kilara, A. (2004). Emulsifying peptides from the tryptic hydrolysis of casein. J. Food Sci. 69, FCT154-FCT163. 

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