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Publisher Summary This chapter describes posttranslational modifications of proteins. A number of short peptides, such as hormones and neuropeptides, are synthesized as multifunctional large polypeptide precursors whose sequences are encoded by mRNA and are assembled by the regular ribosomal synthetic apparatus. There are short polypeptide antibiotics and cell wall constituents that are assembled in step-by-step amino acid activation and condensation catalyzed by specific enzymes in the absence of genetic information and ribosomes. Several of the proteolytic processing steps can be illustrated by a brief review of the biosynthesis of insulin. This disulfide-bonded two-chain structure is encoded as the precursor preproinsulin. It is found that in the early stages of polymerization while the nascent chain is still attached to the membrane-associated polysomes, two cleavages take place. The methylation reactions occur in all prokaryotic and eukaryotic species and involve several different residues such as the side chains of Asp and Glu. It is found that nonmethylated analogs of methylated proteins such as histones, ribosomal proteins, enzymes, binding proteins, and some structural proteins have been produced by mutation or by comparing the same protein produced in different species.

Publisher Summary Biotinylated glycans bound to avidin or streptavidin represent useful glycoprotein models for the study of both glycan processing by Golgi enzymes and glycoprotein interactions with lectins/receptors. The radiolabeled derivative with the 6-carbon extension arm can be prepared by reaction of 6-aminohexanoate with the activated radiolabeled biotin derivative. The product can be further activated with N -hydroxysuccinimide. An alternative for the latter preparation is to modify the α-amino group of the glycopeptide with N -blocked aminohexanoic acid and then, after unblocking, to react with activated biotin. The avidin-bound derivative with the extension arm (biotinamidohexanoyl) shows a slow cleavage under the same conditions. Similarly, exposure of the free and bound biotinylated glycans to α-mannosidase shows significant effects of the protein matrix on both the rate and the products of the mannosidase action. A major area of use for these derivatives has been in the investigation of the effect of the protein matrix on the individual processing enzymes from Golgi membranes.

Publisher Summary Affinity labeling by definition applies to all molecules possessing a site that binds another molecule with a certain degree of specificity and affinity. In biology the proteins are the obvious ligand-binding molecules, and in practice the affinity labeling techniques have primarily involved proteins. The complete understanding of how proteins carry out their biological function requires a detailed understanding of how this binding site is constituted, and this has become the key problem in the area of protein structure-function analysis. The purpose of this chapter is to present a descriptive overview of the current status of affinity labeling; the evolution of the concepts and techniques, the most general picture of the principles involved, the terminology and the criteria commonly applied, and the current and prospective applications of affinity labeling techniques. In this chapter the discussion is kept general and is based on hypothetical examples. The chapter explains the principles and types of reagents, the Covalent Bond formation, interpretations, variations and special considerations and the use of affinity labeling.

Publisher Summary It is most convenient to liberate naturally occurring N-linked oligosaccharides from glycoproteins by exhaustive digestion of the protein with proteases, producing oligosaccharides with a single asparagine residue still attached. Some methods have been devised for derivatizing the Asn as a means of incorporating the oligosaccharide unit into proteins. The subject of this chapter is one such method, based on the direct activation of the Asn moiety by oxidative deamination–decarboxylation with ninhydrin. There is a description of the materials used in the method. A rigid protocol for the reaction was developed because of the instability of the product of the ninhydrin reaction. The main variables tested were temperature and pH in both the activation and the coupling reactions. Under the procedure described in the chapter, it was possible to consistently incorporate 15–20% of the starting oligosaccharide into serum albumin in the reaction of six different ovalbumin-derived oligosaccharide-Asn derivatives. The resulting neoglycoproteins showed several characteristic features of stable glycoproteins in binding to the appropriate lectin and being preferentially cleared from circulation of rats.

Publisher Summary This chapter describes enzymatic hydrolysis of native, soluble proteins and glycoproteins. Protein substrates are denatured to achieve optimal rate of hydrolysis. For native, globular proteins stable at neutral pH, an initial digestion with an acid protease at pH 2-3 significantly facilitates the subsequent digestion with the more commonly used neutral proteases. Cross-links, particularly disulfide bridges, significantly interfere with the digestion of the peptide bonds near the involved cysteine (Cys) residues and also the proteins rich in disulfide bonds generally give incomplete release of amino acids under conditions where disulfide-free proteins appear to be completely hydrolyzed. The co– and posttranslational amino acid derivatives may also seriously interfere with enzyme-catalyzed peptide bond cleavage. The digestion of proteins is assessed without any prior chemical treatment (oxidation, reduction, and heat denaturation). Two groups of substrates are subjected to the digestion protocol, one consisting of common, well characterized proteins with different disulfide content, pancreatic ribonuclease, egg white lysozyme, yeast enolase, and insulin, and the other consisting of glycoproteins with different carbohydrate types and content, ovalbumin, γ -globulin, and submaxillary mucin.

Publisher Summary This chapter describes the active site-specific reagents and transition-state analogs for enolase. There is only one active site-specific reagent known to form a covalent derivative with the enzyme— namely, glycidol phosphate (1,2- epoxipropanol 3-phosphate). In addition, there are pairs of analogs, D-tartronate semialdehyde 2-phosphate and 3-amino enolpyruvate phosphate, which form very tight noncovalent complexes with the active site of enolases. Because of the strong UV absorbance of the complexes, these compounds have been useful in establishing the number of active sites in the enzyme, and as likely transition state analogs, their interaction with the enzyme has shed some light on the mechanism of catalysis. Glycidol phosphate is readily prepared by phosphorylation of commercially available glycidol with POC1 3 , and it satisfies the major requirements for an active site-specific reagent for rabbit muscle enolase. The chapter describes dissociation constants and stoichiometry of enolase-inhibitor complexes, the spectral properties of the enolase inhibitors tartronate semialdehyde phosphate (Ttal 2P) and 3-amino enolpyruvate, and the synthesis of Ttal 2P from commercially available D-gluconolactone. A representation of the complexes of Ttal 2P and Am- e PrvP with the active site of enolase is given as a basis for the comparison with the proposed transition state in the reversible dehydration of glycerate 2-phosphate to enolpyruvate phosphate. It is shown that the enolase-analog complexes form only in the presence of Mg 2+ and that both compounds compete with substrate for the active site support the proposition that the two compounds behave as true substrate (transition state) analogs.

Publisher Summary This chapter discusses a system of complementary cellulose ion exchangers (phosphocellulose and TEAE-cellulose), while emphasizing on peptide separation with a minimum of special equipment for peptide detection. The separation of the 13 tryptic peptides from performic acid-oxidized bovine pancreatic ribonuclease is used in the chapter to illustrate a typical application of phosphocellulose chromatography, gel filtration, and TEAE-cellulose to a typical peptide fractionation using only inorganic buffers and monitoring all chromatograms at 215 nm. There are two dipeptides and one tripeptide in this peptide mixture and these peptides were detected in the case illustrated in the chapter. It is emphasized, however, that the absorbance of the dipeptides is sufficiently low to make dipeptide detection hazardous at best. The particular solvents and gradients used in the separation of ribonuclease peptides are not likely to be universally applicable. It is found that for most peptide mixtures, a considerably higher final KCl concentration (0.5-1.0 M) is required for the elution of all the peptides.

Publisher Summary This chapter focuses on Fabry's disease. Fabry's disease is an X-linked inborn error of glycosphingolipid catabolism. Transplantation of enzymatically active renal tissue into patients with Fabry's disease has resulted in decrease to normal of the accumulated glycosphingolipid substrate in plasma. The diagnosis of hemizygotes and heterozygotes for Fabry's disease can be confirmed by the deficient activity of ceramide trihexosidase in plasma, urine, leukocytes, cultured fibroblasts, or biopsied tissue. The chapter also discusses clinical characteristics of Fabry's disease. The clinical manifestations of the disease result from the generalized visceral deposition of the Fabry glycosphingolipid, particularly in the cardiovascular-renal system. Three separate approaches to enzyme replacement have been utilized in Fabry's disease. These are: (1) the plasma infusion, (2) renal transplantation, and (3) direct enzyme infusion.

Publisher Summary This chapter discusses the mechanism and properties of various bifunctional reagents such as bifunctional maleimide derivatives, alkyl halides, aryl halides, and isocyanates. Three general types of product are considered in the reaction of proteins with bifunctional reagents, each product providing different ways of gathering information about the structure and function of proteins. The types of product and their potential uses are intramolecularly cross-linked proteins, homopolymers, and heteropolymers. The selection of the proper reagent must be determined primarily by the specific product desired. The characterization of the product requires the same procedures as the characterization of proteins modified with monofunctional reagents. Glutaric dialdehyde reagent is commercially available as a 25% aqueous solution and can be purified by recrystallization as the bisulfite addition compound. The synthesis of a number of bifunctional imidoesters is described and preliminary results on their reaction with proteins are available. Bifunctional acylating reagents include a wide variety of compounds of different dimensions and reactivities, with monofunctional analogs available for control experiments.

Publisher Summary This chapter discusses the use of bifunctional reagents to proteins, and several cross-linking reagents, such as mustards and epoxides, which have recently been used to cross-link DNA. It has been demonstrated that at least some of the cross-links join opposite strands of the reacted DNA, therefore, very interesting structural and functional properties of double-stranded nucleic acids may be explored by the use of bifunctional reagents. In the reaction of proteins with bifunctional reagents, three general types of product can be considered, each product providing different ways of gathering information about the structure and function of proteins. The types of product––intramolecularly cross-linked proteins, intermolecularly cross-linked proteins (homopolymers), and intermolecularly cross-linked protein complexes, composed of different proteins (heteropolymers) or of proteins and other reactive molecules and their potential uses are discussed in the chapter. N-substituted bismaleimide derivatives provide highly specific and mild bifunctional reagents. Bifunctional reagents is used in the study of the sub-unit structure of oligomeric proteins.