Christian B. Anfinsen

[31] Affinity chromatography

Publisher Summary The selective isolation and purification of enzymes and other biologically important macromolecules by “affinity chromatography” exploits the unique biological property of the proteins or polypeptides to bind ligands specifically and reversibly. Affinity chromatography exploits the phenomenon of specific biological interaction in a large variety of protein–ligand systems. A solution containing the macromolecule to be purified is passed through a column containing an insoluble polymer or gel to which a specific competitive inhibitor or other ligand has been covalently attached. Proteins not exhibiting appreciable affinity for the ligand pass unretarded through the column, whereas those which recognize the inhibitor are retarded in proportion to the affinity existing under the experimental conditions. The specifically adsorbed protein can be eluted by altering the composition of the solvent so that dissociation occurs. Affinity chromatography may be useful in concentrating dilute solutions of proteins, in removing denatured forms of a purified protein, and in the separation and resolution of protein components resulting from specific chemical modifications of purified proteins. Inherent advantages of this method of purification are the rapidity and ease of a potentially single-step procedure, the rapid separation of the protein to be purified from inhibitors and destructive contaminants, such as proteases, and protection from denaturation during purification by active site ligand-stabilization of protein tertiary structure.

The correlation of ribonuclease activity with specific aspects of tertiary structure

Various physical parameters of bovine pancreatic ribonuclease and of some of its derivatives prepared by oxidation or reduction of disulfide bridges, methylation, and proteolytic digestion, have been investigated. Ultraviolet spectrophotometric measurements appear to establish a correlation between specific spectral properties of the materials and their enzymic activity. Viscometric and optical rotary studies, on the other hand, indicate that minor modifications in secondary structure may occur without detectable inactivation. Polyvalent anions and polyanions almost completely prevent the unfolding and spectral shift effect of 8 M urea, and of guanidium ions at concentrations between 1 and 3 M. These findings suggest that the full activity of ribonuclease, in the presence of such denaturing agents, is due to a refolding of the protein under the influence of ribonucleic acid.

The reductive cleavage of disulfide bonds and its application to problems of protein structure

Abstract The quantitative reduction of disulfide bonds in ribonuclease and lysozyme may be achieved at room temperature with thioglycolic acid in 8 M urea at pH 8. 5 . Sulfhydryl groups of the resulting cysteine residues may be reacted with iodoacetic acid to yield the S-carboxymethyl derivatives of these residues. The S-carboxymethylcysteine content of reduced and alkylated proteins may be estimated by dinitrophenylation of acid hydrolysates followed by chromatographic isolation of DNP-SCMC. Under the conditions of reduction and alkylation employed, no significant modification, stable to the conditions of hydrolysis employed, of tyrosine, tryptophan, lysine, or histidine residues occurs. The method described is, therefore, of particular value as an initial step in the study of amino acid sequence in such tryptophan-rich proteins as lysozyme. The digestion of reduced and alkylated lysozyme and ribonuclease with trypsin proceeds rapidly and yields, in each case, a population of peptide fragments which is in good agreement with that to be expected from the lysine and arginine contents of these proteins.

Protein Structure in Relation to Function and Biosynthesis

Publisher Summary The chapter discusses the protein structure in relation to function and biosynthesis. The chapter presents the outline of strategy that summarizes the approaches to sequence determination most generally followed. The sequence of individual protein chains appears to be best studied by the isolation and characterization of fragments produced by proteolytic digestion. The chapter explores the partial hydrolysis of proteins. Partial acid hydrolysis is probably useful mainly for the subsequent study of the larger peptide fragments produced by proteolysis. Sequence studies on proteins and peptides are dependent upon the isolation and characterization of residues or small groups of residues occupying definite positions in the peptide chain. The two unique residues in a peptide (except in cyclic compounds such as gramicidin) are those possessing a free amino group (the N -terminal residue) and those possessing a free α-carboxyl group (the C -terminal residue). These can be identified by suitable labeling techniques or by enzymatic methods. The determination of the nature of disulfide bridges in the insulin molecule has been described. Methods for the production and isolation of these bridges from proteins are quite empirical, and their successful application depends to a large degree on the chemical nature of the molecule under investigation. The approximate assignment of amide nitrogen to specific dicarboxylic amino acids in the sequence of a protein chain may be made on the basis of direct analyses for ammonia in hydrolyzates of peptides isolated from partial digests of such chains. This approach to the problem is a laborious one and may often not permit unequivocal assignments. The structure of individual proteins is also discussed.

An immunological approach to the conformational equilibrium of staphylococcal nuclease

Abstract The conformational equilibrium constant, K conf , of staphylococcal nuclease, describing the equilibrium between the native conformation and non-native or disordered conformations, has been estimated using an immunologic method and an interpretive model. Using goat antisera prepared toward a conformationally disordered nuclease fragment (99–149), antibodies specific for the disordered form of the helix-rich sequence 99 to 126, anti-(99–126) R , were isolated by sequential immunoabsorption. Anti-(99–126) R forms soluble 7 S complexes with fragment (99–149), but this interaction may be inhibited by a large excess of nuclease. By using fragment (99–149) preferentially carbamylated at the α-amino terminus with KN 14 CO and rabbit anti-goat immunoglobulin to distinguish between antibody-bound and free fragment (99–149), an assay for the quantitation of the degree of inhibition of anti-(99–126) R . (99–149) complex formation by nuclease was developed. Using a formal analysis based on the hypothesis that nuclease is in a conformational equilibrium between a folded and unfolded form and that anti-(99–126) R binds effectively only to the unfolded form, the K conf of nuclease was estimated to be 2900. In the presence of the ligands Ca(II), or Ca(II) and thymidine-3′,5′-diphosphate, K conf values of 6500 and 30,000 to 50,000 were estimated, respectively. The K conf of nuclease at 4 °C and 39 °C was 3900 and 400, respectively.

Folding of staphylococcal nuclease: Kinetic studies of two processes in acid renaturation

Abstract The folding of acidified staphylococcal nuclease, upon neutralization, was studied in a stopped-flow spectrofluorometer by measuring the increase in tryptophanyl fluorescence during renaturation. At 25 °C and 0.1 ionic strength, the fluorescence change may be described by two first-order rate processes with half-times of 55 and 350 msec. No significant change in either rate was effected on varying the initial pH from 3.2 to 3.8 (corresponding to 0 to 50% levels of folding) or the initial ionic strength from 0.001 to 0.1. In contrast, the over-all rate of folding is dependent upon temperature. The half-time of the slower rate process decreases from 600 msec at 13 °C to 150 msec at 38 °C; the half-time of the faster rate process does not change significantly over this temperature range. These results suggest that the two processes may correspond to a sequence involving nucleation of ordered structure followed by the formation of hydrophobic interactions. Possible structural correlates of these processes are discussed with respect to the crystallographic model of this protein.

8 Staphylococcal Nuclease, Chemical Properties and Catalysis

Publisher Summary This chapter discusses chemical properties and catalysis of staphylococcal nuclease. Staphylococcal nuclease yields 3′-nucleotides upon hydrolysis of polynucleotide chains. This feature of its catalytic action was of considerable use in the classic studies of Kornberg and his collaborators in the determination of nearest neighbor base frequencies in synthetic deoxyribonucleic acid (DNA). A radioactive phosphorus atom esterified to the 5′-OH group of deoxy- ATP—for example, would be located, after staphylococcal nuclease digestion of the newly synthesized DNA, on the 3′-mononucleotide, dXp, derived from the sequence dXpdA-. The enzyme has also been used extensively in studies on the elucidation of sequences of polynucleotidc chains. In addition to interest in this enzyme because of its catalytic characteristics, a considerable body of information has accumulated on staphylococcal nuclease as a protein molecule. Its relatively small size, the absence of covalent cross-linkages, and its behavior upon binding a variety of ligands have made it an ideal model substance for the study of various aspects of protein chemistry including X-ray crystallography. These investigations are reviewed in the chapter.

Rapid solid-phase synthesis of bradykinin

Abstract Modifications of the Merrifield solid phase method for peptide synthesis are presented. The technique as developed has permitted the synthesis of bradykinin, in good yield and high purity, in less than five hours.

A method for the specific proteolytic cleavage of protein chains

Abstract Oxidized ribonuclease was reacted with carbobenzoxychloride, the carbobenzoxylated protein obtained was digested with trypsin, and the carbobenzoxy groups were removed from the tryptic digest by means of anhydrous hydrogen bromide. The analysis of the tryptic digest before and after decarbobenzoxylation showed that four new end groups were liberated during trypsin digestion, in accordance with the presence in ribonuclease of four arginine residues. The peptide fragments formed were separated by high-voltage paper electrophoresis. The experiments described suggest that this method may be of general use as a preliminary step in the determination of the sequential arrangement of amino acid residues in peptidic chains.

Fractionation of antibodies against staphylococcal nuclease on 'sepharose' immunoadsorbents

IMMUNIZATION of an animal with a protein antigen usually elicits a mixture of serum antibodies against different determinants on the immunizing protein1. The correlation of amino-acid sequence and three-dimensional conformation of the antigen with its immunochemical behaviour has been facilitated by the use of specific immunoadsorbents. In this technique, the antigen is coupled chemically to an insoluble support, such as bromoacetyl cellulose2,3 and an ethylene–maleic anhydride copolymer4 used in studies of antigenic sites in lysozyme5 and myoglobin6. ‘Sephadex’ and, more recently, ‘Sepharose’ have been introduced as effective supports for attachment of antibodies7, hormones8, enzyme substrates9 and polypeptide fragments10.