Serge N. Timasheff

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.

Hydrogen ion equilibria of the genetic variants of bovine β-lactoglobulin

Abstract The hydrogen ion equilibria of β-lactoglobulins A, B, and C have been examined. The resulting titration curves can be accounted for in terms of the normal ionization of all groups, with the exception of two histidines in the C variant and two carboxyls in all three of the variants. Conformational changes at pH 4.5–6 and 6.5–9 release these abnormal groups to ionization.

The extrapolation of light-scattering data to zero concentration

The problem of protein interactions has been a focus of research over a long period of time. It is well known that such systems as enzyme-substrate and antigen-antibody interactions involve the association of the active protein with other molecules. Furthermore, the degree of molecular dispersion of a protein may greatly affect its biological properties as shown, for example, by Nord and co-workers (1-5). Bier and Nord (5, 6) were among the first to show that light scattering could be used for the study of protein interactions. In general, this technique has gained wide acceptance for the study of protein associations and the thermodynamics of macromolecular interacting systems (7-17). The interactions which a protein undergoes in solution, however, lead often to serious complications in the analysis of light-scattering data. These complications usually manifest themselves in rendering the extrapolation to zero protein concentration very difficult or in introducing a certain amount of uncertainty into the meaning of the intercept. As a result, a certain amount of confusion has existed as to whether the correct molecular weight of polyelectrolytes, such as proteins, can be obtained only in the presence of salt or, to the contrary, only in the salt-free case. It is the purpose of this paper to present an analysis of various factors affecting the extrapolation of light-scattering data to zero concentration and to outline by typical examples the proper procedure to be used in various cases.

Optical rotatory dispersion and circular dichroism properties of yeast mitochondrial DNA's.

Abstract Optical rotatory dispersion and circular dichroism spectra of mitochondrial DNAs from two “grande” and three cytoplasmic “petite” strains of Saccharomyces cerevisiae were determined. The optical rotatory dispersion spectra showed a blue shift compared to those of control DNAs; furthermore, they exhibited a shoulder at 270 mμ. The Same-jima-Yang relationship between [α] and base composition was not followed. In the circular dichroism spectra, the broad maximum at 276 to 278 mμ exhibited by control DNAs was replaced by a peak at 273 mμ and two shoulders at 263 and 281 mμ, respectively. The shoulder at 263 mμ had a higher intensity in the DNAs derived from the petite mutants compared to those prepared from the grande strains. In both optical rotatory dispersion and circular dichroism spectra, the negative bands, at 255 and 247 mμ, respectively, were greatly increased in intensity. A comparison of the spectra of yeast mitochondrial DNAs with those of poly(dAT:dAT) and poly(dA:dT) showed that the peculiar features of the yeast mitochondrial DNA spectra may be due to contributions from both alternating poly(dAT:dAT) and non-alternating poly(dA:dT) structures.

Association of αs-casein C in the alkaline pH range

Abstract The molecular weight of the α s -casein C monomer was established as 26,900 by light-scattering experiments in anhydrous formic acid, chloroethanol, and 26% aqueous methanol. In 56% aqueous methanol, this protein exists in the form of a trimer. The state of association of α s -casein C has been studied at room temperature in aqueous solutions in the pH range of 8.0–9.5 and at ionic strengths between 0.02 and 0.30 (NaCl). The light-scattering and sedimentation data are characteristic of a rapidly reequilibrating system at ionic strengths of 0.1 and greater; the predominant species are dimer, tetramer, and hexamer. Lowering the ionic strength to 0.02 caused dissociation to monomer, the reequilibration being slow. Dissociation is favored by an increase in pH and a decrease in ionic strength. The variation of a number of properties, such as viscosity, sedimentation, and optical rotatory dispersion, suggest that α s -casein C has a structure more flexible than that of native globular proteins; the molecule, however, is much less flexible than those of “denatured” proteins, e.g., globular proteins in structure-breaking solvents.

Studies on acid deoxyribonuclease: VII. Conformation of three nucleases in solution☆☆☆

Abstract The optical rotatory dispersion, circular dichroism, and infrared spectra of acid DNase, pancreatic DNase, and Staphylococcus aureus nuclease have been determined. The conformations of these three nucleases differ greatly from each other and from that of ribonuclease; while the acid and pancreatic DNases contain little α-helix, the bacterial enzyme has ca . 25% of that conformation; acid DNase probably contains antiparallel pleated sheet β-structure.

Infrared titration of lysozyme carboxyls

Abstract The dissociation of carboxylic groups in lysozyme was measured as a function of pH by differential infrared absorption spectroscopy. Analysis of both the 1707 cm −1 (COOH) and the 1565 cm −1 (COO − ) bands showed that of the 10 ionizable groups, two have highly abnormal p K values, 2.0 and 6.5, while the others vary between 3.5 and 5.5. Assignments to specific residues in the amino acid sequence are made in terms of known features of their microenvironment.

On the mechanism of α-chymotrypsin dimerization

Abstract The dimerization of α-chymotrypsin between pH 2.5 and 8 has been examined in terms of electrostatic interactions. In appears that the association involves the attraction between a pair of groups with p K values of 3.3 and 8.5 and the repulsion between a pair of carboxyls with p K 5.0.

On the electrophoretic heterogeneity of trypsin

Abstract Two electrophoretic components of trypsin were separated and shown to possess comparable proteolytic activity on natural and synthetic substrates.

The role of tyrosines in elastase

Abstract The conformational behavior of elastase between pH 7 and 11 has been investigated, with particular emphasis on tyrosine residues. Of these, ten titrate normally and one is totally inaccessible to ionization. On the basis of chemical modification, the eleven tyrosines can be subdivided into three reactive classes and one totally unreactive. The first class, consisting of eight groups, can be cyanurated and is easy to acetylate; the other two classes consist of one group each, which can be acetylated with increasing difficulty but cannot be cyanurated. The eleventh residue is inaccessible to either reagent. Circular dichroism studies showed that the secondary structure remains invariant between pH 5 and 11, since the far uv CD spectrum remains unchanged. The fact that acetylation of ten tyrosines neither affects the far uv CD spectrum nor the enzymatic activity rules out the structural stabilization of the active enzyme by a tyrosine. Circular dichroism studies at higher wavelengths indicate that the alkaline transitions found in chymotrypsin do not occur in elastase.