Robert Townend

The state of amino acid residues in β-lactoglobulin

Abstract The extent of availability of the tryptophan, cysteine, and tyrosine residues of β-lactoglobulin has been examined under various conditions, using techniques of difference spectroscopy and chemical modification. It has been found that the solvent perturbation difference spectra of the tryptophans are independent of the state of association of this protein (monomer, dimer, octamer). Modification of the single cysteine residue per subunit affects the octamer formation to different extents, whether the derivative is neutral or carries a positive or negative charge. Of the four tyrosines per chain, two are readily available, one is hindered, and the fourth totally unavailable. These results are analyzed in terms of the known structural features of the protein, as well as of the various association reactions and conformational changes which it undergoes.

[10] Light scattering and differential refractometry

Publisher Summary This chapter presents the basic principles to discuss the problems and to describe the practical techniques involved in performing light scattering and differential refractometry measurements on multicomponent systems and to describe some of the instrumentation available for angular measurements. Among the techniques available for the characterization of macromolecules in solution, a particularly useful one is light scattering. With this method, it is possible to determine at relatively low protein concentration the molecular weight, the degree of association, interactions with solvent components, and, if the macromolecule is large enough, its size and general shape. When the dimensions of a particle become comparable to the wavelength of the incident radiation, interference occurs among the radiations scattered from individual elements within the particle. In the case of light scattering, this effect becomes significant when the maximal dimension of a particle becomes of the order of λ/10. As the wavelength of the incident radiation is of the order of 4000A, this means that particle dimensions must attain 400A to be resolved by light scattering.

The isolation and amino acid composition of two peptides from chymotryptic digests of β-lactoglobulins A and B

Abstract Short-term chymotryptic digests have been carried out with β-lactoglobulins A and B, performic acid-oxidized and S-sulfonated derivatives. Differences in the peptide patterns have been found after high-voltage electrophoresis only when disulfide bonds were previously cleaved. Two peptides have been isolated from chymotryptic digests of S-sulfonated β-lactoglobulins, and their amino acid compositions have been determined to be as follows: For the peptide from the β-A derivative: Asp 2 , Thr 1 , Glu 4 , Pro 1 , Ala 3 , Val 1 , Ileu 2 , Phe 1 , 1 2 Cys 1 , Lys 4 ; and for the peptide from β-B: Asp 1 , Thr 1 , Glu 4 , Pro 1 , Ala 3 , Val 1 , Ileu 2 , Phe 1 , 1 2 Cys 1 , Lys 4 , Gly 1 . The sole difference lies in the aspartic acid-glycine content. The valine-alanine difference in β-lactoglobulins A and B remains unaccounted for in the present study.

β-Lactoglobulins A and B: The environment of the asp/gly difference residue☆

Abstract The environment of the aspartic acid-glycine substitution has been examined. A pair of peptides of differing mobility, showing the known asp-gly substitution of the native variants, has been isolated from tryptic digests of S -sulfonated β-lactoglobulins A and B. The amino acid composition of the peptides shows a large concentration of carboxylic residues in the neighborhood of the difference amino acid. Comparison with the physicochemical properties of the variants indicates that this area of the primary structure is probably implicated in the low-temperature aggregation, since differences between the two species in degrees of this reaction correlate with the density of carboxyl groups in the vicinity of the substitution.

Molecular weight of some human and cow caseins

Abstract The molecular weights of a number of caseins isolated from human and cow milks have been determined by sedimentation equilibrium. The predominant human caseins, which differ only in their content of phosphorus, have a molecular weight of approximately 25,000 daltons, a value similar to that of cow β- and γ-caseins. Other cow caseins (R, S, and TS) have molecular weights of 13,000–16,000. All values obtained by these measurements agree with minimum molecular weights calculated from content of tryptophan assuming the presence of one residue per molecule.

Physicochemical comparison of goat β-lactoglobulin with the bovine varieties

Abstract β-Lactoglobulin was prepared from mixed goat milk and multiple amino acid analyses were carried out. One difference in basic residues was found from the analysis previously reported. Ultracentrifugal, light-scattering, and optical rotatory dispersion experiments showed that the goat protein is quite similar to the bovine β-lactoglobulins in most of its physical properties, but indications were obtained that it is conformationally less stable at low pH values than its bovine analog. Titration experiments indicate that six of the free carboxylic acid (Asp and Glu) residues in monomeric bovine β-lactoglobulin B do not occur in titratable form in the caprid β-lactoglobulin. These experiments also indicate a low pH-induced conformational change to a molecule of slightly greater radius.

β-Lactoglobulin As A Model of Subunit Enzymes†

Summary The states of association of the β-lactoglobulins are discussed, with particular emphasis on the mechanism of the pH 4–5 low temperature octamer formation. The bell-shaped pH dependence of this reaction is explained, linking it to a small conformational change observed in the same pH range. The physico-chemical properties of this protein are examined in terms of the allosteric model and it is proposed that β-lactoglobulin is a good model of subunit allosteric enzyme systems.

Amino acid analysis of the β-lactoglobulins from individual cows of two phenotypes

Abstract Amino acid analyses were performed on β-lactoglobulin samples prepared from individual dairy cows, 24 of which were homozygous for β-lactoglobulin A and 9 for β-lactoglobulin B. No differences were found in individual contents of any amino acids other than the previously known β-A-β-B difference amino acids. The total analyses agreed well with previously published analyses on β-A and β-B obtained from pooled milk of many animals.

Studies on acid deoxyribonuclease: X. Molecular weight in denaturing solvents

Abstract Hog spleen acid DNase, an enzyme which peptide mapping indicates to be formed by two similar or identical subunits, has been subjected to experiments designed to determine the molecular weight of the monomeric subunit. The molecular weight as determined at low pH and in the presence of 6 m guanidine, with or without added β-mercaptoethanol, was found to be very close to that of the native enzyme molecule. It is concluded that the enzyme molecule cannot be dissociated into subunits under a variety of conditions which are extremely effective with other proteins.