Biochimica et Biophysica Acta (BBA) - Protein Structure最新文献

Thermoplasma acidophilum is a mycoplasma-like thermophilic organism that has been classified with the Archaebacteria. It has a single superoxide dismutase (superoxide : superoxide oxidoreductase, EC 1.15.1.1) which is composed of four identically sized subunits. It has a metal content per molecule of two atoms of iron and probably one of zinc and a molecular weight of 82 000. The amino acid composition is rich in tryptophan and is typical of the manganese or iron superoxide dismutases found in other prokaryotes. However, the enzyme is resistant to denaturation by chloroform plus ethanol, by sodium dodecyl sulfate plus urea or by heat. In these respects it resembles the copper-zinc superoxide dismutase of eukaryotes. It is suggested that the enzyme may belong to a new group of superoxide dismutases.

Serum albumin, which itself has a surface tension of ⋍70.3 erg/cm², when dissolved in water lowers the surface tension of water from 72.5 to ⋍50 erg/cm², as measured by a variety of means, including the pendant drop, the Wilhelmy plate and the platinum ring methods. Equally low and even lower surface tensions are found with the contact angle method, on a thin layer of albumin that had been adsorbed onto a low energy surface and subsequently exposed to air. Surface tensions of drops of albumin solutions varying in concentration from 0.01 to 5.5% (w/v) yielded, with a contact angle method, values that only varied between 67 and 61 erg/cm². With the pendant drop, the Wilhelmy plate and the platinum ring methods, one essentially measures the surface tension at the air-liquid interface, at which proteins tend to adsorb, and where reversible or irreversible reorientation can be expected. The same holds for a thin layer of protein adsorbed onto a low energy surface, exposed to air. Thus, when through the very act of surface tension measurement, or after adsorbing protein onto a substrate, protein is exposed at the air-liquid interface, it apparently loses the pronounced hydrophilicity characteristic of its native hydrated state and manifests through reorientation a much more hydrophobic tertiary configuration.

The complete amino acid sequence of 87 residues has been determined for the N-terminal CNBr fragment of Tachypleus tridentatus hemocyanin α chain. It is rich in histidine and contains one free cysteine. The N-terminal sequence of 20 residues shows homologies with other arthropod hemocyanins.

The fast step in the conformational change of human serum albumin from the alkaline to the neutral form of the albumin-bilirubin complex is studied by various pH jump experiments in a stopped-flow apparatus. The results indicate that the fast step is caused by electrostatic attraction between a carboxylate group of bilirubin and a histidine residue of albumin.

Trypsin digestion of haptoglobin resulted in four glycopeptides. The glycopeptides were characterized by amino acid composition and molecular weight, as determined by thin-layer chromatography, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. Hemoglobin-binding capacity and immunological properties were investigated. Glycopeptides I and II did not form an active complex with hemoglobin and they inhibited the reaction of haptoglobin with specific antiserum by over 70%. Glycopeptides III and IV showed 11 and 4% of the hemoglobin-binding capacity and 82 and 67% of antigenic reactivity of native haptoglobin, respectively. Glycopeptide IV contained three antigenic determinants, whereas glycopeptides III contained four, one of them being exposed by trypsin digestion. In crossed two-dimensional immunoelectrophoresis, glycopeptide III showed at least four components reacting with antihaptoglobin serum, and glycopeptide IV, two components.

The desorption patterns of serum proteins in hydrophobic chromatography suggest that serum proteins that remain immersed in an aqueous medium and do not become involved in a protein-air interface are very hydrophilic. Contact angle measurements on fairly thick layers of hydrated serum proteins, formed on ultrafiltration membranes, yield surface tensions that correlate well with the degree of hydrophilicity derived from desorption data obtained by hydrophobic chromatography. For further confirmation the absorptivity of four human serum proteins was measured with respect to surfaces of different polymers of various surface tensions, from solution in aqueous solvents of different surface tensions. The surface tension of the solvent from which a dissolved protein adsorbs to precisely the same extent onto all solid substrates (regardless of their surface tensions) is equal to the surface tension of that protein. The surface tensions found by the contact angle (first value given) and by the protein adsorption methods (second value given) were, in erg/cm²; α₂-macroglobulin, 71.0, 71.0; serum albumin, 70.5, 70.2; immunoglobulin M, 69.5, 69.4; immunoglobulin G, 67.4, 67.7.

Purified antithrombin III has been reported to have bound glucocerebroside, the major glycolipid of plasma. We have separated whole plasma by ultracentrifugation into lipoprotein-rich and lipid-deficient fractions and demonstrated that glucocerebroside and antithrombin III clearly separate into different fractions. Antithrombin III does not have glucocerebroside associated with it.

A photoactivatable fluorescent anthraniloyl group has been directed to the active-site serine group of α-chymotrypsin and trypsin. The acylated derivatives are nonfluorescent until irradiated. When activated by light a highly reactive nitrene is generated which is capable of covalent insertion into the protein matrix. The resultant insertion product of this photolysis is a highly fluorescent reporter group which has little rotational mobility and is cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked enzyme and deacylated enzyme were determined. These measurements show that little relaxation occurs on the nanosecond time scale for the cross-linked enzyme, but upon deacylation of the serine increased dipolar relaxation of the protein with the attached reporter group is observed. The use of these active-site directed photoactivatable fluorescent probes can be extended to probe the active-site structure of complex enzymes and conformational dynamics of active-site regions in proteins and to serve as potential functional site labels in fluorescence resonance energy transfer measurements.

The conformational properties of the protease from Staphylococcus aureus strain V8 were studied by the CD probe. The CD spectra in the far ultraviolet zone displayed a negative band at 205–207 nm but no positive bands were observed at 191–198 nm. This indicates that the protease was devoid of significant amounts of the α-helix and pleated sheet conformations, i.e., that the polypeptide chain was folded into a unique irregular (aperiodic) conformation. The structure was relatively insensitive to sodium dodecyl sulfate and high concentrations of aliphatic alcohols but it was readily perturbed by acid and alkali. This suggests that the three-dimensional structure of this protein is stabilized chiefly by electrostatic interactions. Significant differences in the tertiary structure of the protease were indicated by the CD spectra at the two enzyme activity maxima (pH 4.1 and pH 7.6–8.2).