Finn Wold

[1] Affinity labeling—An overview

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.

Enolase: Multiple Molecular Forms in Fish Muscle

Starch-gel electrophoresis shows three distinct molecular forms of enolase in each of eight different species of Salmonidae. The three enolases do not appear to be artifacts of isolation, and their electrophoretic patterns are completely reproducible. The patterns are also highly characteristic for each individual species of fish, and together with the overall myogen patterns they represent unequivocal means of taxonomical identification.

Comparative studies on structural and catalytic properties of enolases

Abstract Enolase has been partially purified from dog, beef, mouse, frog, turtle, ocean perch, snails, soybeans, oats, and Leuconostoc mesenteroides, using procedures similar to that used to prepare enolase from rabbit muscle. Pure enolases from yeast, rabbit muscle, rainbow trout, chum salmon, and coho salmon were also included in this comparative study. The catalytic properties of all the enolases studied have been found to be very similar. The Km values fall within the range of 0.40–1.58 × 10−4 m . All the enzymes require Mg2+ for activity and are inhibited by F− with similar inhibition constants. pH optima can be divided into two classes, with the pH optimum of vertebrates at pH 7.0 ± 0.1, and the optimum for yeast and higher plants at pH 7.6–8.0. The molecular weight, as estimated by sedimentation in sucrose density gradients, is quite similar for the enolases studied. On the other hand, relative electrophoretic mobility and immunochemical studies revealed considerable variation in the other structural properties. We tentatively conclude from the data that the active site of enolase expressed by widely different protein molecules in different species has remained remarkably constant through biochemical evolution.

Purification and characterization of enolases from coho (Oncorhynchus kisutch) and chum (Oncorhynchus keta) salmon

Abstract Enolase from two species of Pacific salmon has been isolated and characterized. Both crystalline preparations show the presence of three electrophoretically distinct enzyme forms, but while the chum salmon enolases appear in an approximate ratio of 1:3:4, the coho salmon has one predominant (95%) form and only traces of two other enolases. The enolases from the two salmon species have many properties in common. Both have a molecular weight of 100,000 and based on carboxylterminal analyses, contain two polypeptide chains per mole. In both enzymes both chains terminate with an isoleucine-asparagine sequence. Other points of structural similarity are illustrated by the identical analyses of 12 cysteines and 1 cystine for each of the two enzymes, and their very similar amino acid content. Major differences are found in their different electrophoretic behavior and in the tryptophan content. The kinetic parameters ( K m V max , pH-optimum, Mg 2+ activation, and fluoride inhibition) are essentially identical for the two enzymes.

A new chemical synthesis of D-tartronic acid semialdehyde phosphate and D-glyceric acid 2-phosphate

Abstract A short synthetic route for the preparation of D -tartronic acid semialdehyde phosphate and D -glyceric acid 2-phosphate from the readily available starting material, D -gluconolactone, is described. Enzymic tests of the two phosphate esters as inhibitor and substrate, of enolase, respectively, are reported.

D-ERYTHRONIC ACID 3-PHOSPHATE. A SUBSTRATE FOR ENOLASE.

Abstract The synthesis of d -erythronic acid 3-phosphate is described. Its activity as a substrate for enolase is demonstrated and discussed.

[77] Complex neoglycoproteins

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.

Chapter 2 – Posttranslational Modifications

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.

[31] Attachment of oligosaccharide-asparagine derivatives to proteins: Activation of asparagine with ninhydrin and coupling to protein by reductive amination

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.

[23] Separation of peptides on phosphocellulose and other cellulose ion exchangers

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.