Modification of Proteins and Peptides

Separation of p-nitrophenol from the internal standard, 2,4-dinitroaniline, is achieved on LiChrosorb RP-18 column (4.6 mm x 250 mm, 10 /urn) from Perkin-Elmer. The mobile phase was a 55 45 (v/v) mixture of water and acetonitrile. The effluent was monitored at 300 nm. 

Lipase was assayed in a final volume of 1.4 mL containing 1.2 mL of substrate suspension [8 mg of p-nitrophenyllaurate dissolved in 10 mL of acetone, which was then added with agitation to 50 mL of 0.2 M phosphate buffer (pH 7.4), 10 mL of 25 mAf sodium taurocholate, 10 mL of 70 mM NaCl, and 20 mL of water] and enzyme in 0.1 M phosphate buffer (pH 7.4). This mixture was incubated at 37°C for 30 minutes before the reaction was stopped by adding 0.1 mL of 5 M HQ. Then 100 fiL of internal standard (0.2 mg/mL of 2,4-dinitroaniline in ethanol) was added. After filtration, samples are injected into the HPLC. The assay is linear with up to 2.5 fig protein and 30 minutes incubation. 

The enzyme source was a commerical pharmaceutical preparation from pancreas of pig. 

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McCracken, P. G. Bolton, J. L. Thatcher, G. R. J. Covalent modification of proteins and peptides by the quinone methide from 2-ZerZ-butyl-4,6-dimethylphenol selectivity and reactivity with respect to competitive hydration, j. Org. Chem. 1997, 62, 1820-1825. 

K. Mizutani, T. Electronic and structural requirements for metabolic activation of butylated hydroxytoluene analogs to their quinone methides, intermediates responsible for lung toxicity in mice. Biol. Pharm. Bull. 1997, 20, 571-573. (c) McCracken, P. G. Bolton, J. L. Thatcher, G. R. J. Covalent modification of proteins and peptides by the quinone methide from 2-rm-butyl-4,6-dimethylphenol selectivity and reactivity with respect to competitive hydration. J. Org. Chem. 1997, 62, 1820-1825. (d) Reed, M. Thompson, D. C. Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di- m-butyl-4-methylphenol (BHT). Chem. Res. Toxicol. 1997, 10, 1109-1117. (e) Lewis, M. A. Yoerg, D. G. Bolton, J. L. Thompson, J. Alkylation of 2 -deoxynucleosides and DNA by quinone methides derived from 2,6-di- m-butyl-4-methylphenol. Chem. Res. Toxicol. 1996, 9, 1368-1374. 

Another form of post-translational modification that may add carbohydrate to a polypeptide is non-enzymatic glycation. This reaction occurs between the reducing ends of sugar molecules and the amino groups of proteins and peptides. See Section 2.1 in this chapter for further details and the reaction sequence behind this modification. 

Other modifications of capillary electrophoresis techniques, described below, have been adopted for the improvement of separation of several substances, including proteins, peptides and aminoacids. Furthermore, new procedures are also being developed to avoid the adsorption of proteins and peptides onto the walls of the capillary and consequently improving their selectivities. 

Modification of the prcformulation format for biotechnological products from the original guidance must be considered. The sections regarding chemical structure, physicochemical properties, and stability may be revised according to the nature and characteristics of proteins and peptides. Aside from the conventional analytical instruments and techniques used in the study of small molecules, methods such as amino acid analysis, sequence analysis bioassay, immunoassay, and enzymatic assay are commonly used and should be included in the report. 

The biosynthetic production process and the resulting product may include several molecular entities or variants originating from post-translational modifications in case of proteins and peptides. These are obtained from recombinant or non-recom-binant cell cultures, and are considered to be part of the product. Individual and possibly collective acceptance criteria must be defined which may take into account mea-... 

This chapter deals with protein structure-function relationships resulting from proteolytic modification, covalent amino acid incorporation, and the effects of these enzymatic modifications on sensory and nutritional quality, on the functional properties, and on the biological activities of proteins and peptides. 

Chymotrypm, probably the most thorou y studied enzyme because of its stability and availability, primarily catalyzes the hydrolysis of amide bonds of proteins and peptides adjacent to the carbonyl group of the aromatic L-amino acid residues of tryptophan, tyro e, and phenylalanine. Therefore, the hydrophobic interaction between the active site and substrate molecules is believed to make a mryor contribution to the stabflity of enzyme-substrate complexes. In fact, the X-ray data showed that the a-chymotryfsin molecule is an elipsoid (51x40x40 A) and that the active site is a hydrophobic cavity of 10—12 Ax5.5 —6.5 Ax3.5 —4.5 A (5). In chemical modification experiments, histidyl imidazole 2, and seryl hydroxyl 1 groups were found to be directly involved in the catal) ic process. The formation of acyl enzyme intermediates at the seryl residue was demonstrated by phyacal and chemical means. 

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