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PubMed1975

Chemical modification of yeast 3-phosphoglycerate kinase.

Sulfhydryl reagents, as well as mild hydrogen peroxide oxidation, do not inhibit the activity of yeast phosphoglycerate kinase, indicating that the single thiol group and 3 methionine residues present in the enzyme are not essential for activity. Nitration of phosphoglycerate kinase by tetranitromethane inhibits the enzyme by reaction with a single tyrosine residue. Substrates provide partial protection against inactivation by nitration. Circular dichroism spectra indicate that no conformational changes occur upon nitration. However, perturbation of the microenvironment surrounding the aromatic amino acid residues, particularly tyrosine, was observed. The same perturbation was observed on addition of the substrate 3-phosphoglycerate kinase to native phosphoglycerate kinase. The role of lysine in the action of yeast phosphoglycerate kinase has been studied by modification with O-methylisourea, 2-methoxy-5-nitrotropone, and pyridoxal phosphate. Guanidination shows that there are lysines essential for phosphoglycerate kinase; extrapolation to zero activity indicates that there are three essential lysines as judged by nitrotroponylation and three essential lysines when the enzyme is reacted with pyridoxal phosphate. Substrates afford partial protection and extrapolation to total protection indicates that up to three lysines are protected by MgITP and one lysine by 3-phosphoglycerate. Spectrofluorescence and optical rotatory dispersion measurements show that there is no detectable conformational change for the guanidinated phosphoglycerate kinase and that there are slight changes in the spectra suggesting that there may be slight conformational changes for the nitrotroponylated and the pyridoxal phosphate-modified enzymes.

Amino Acid Sequence↗
PubMed1975

Properties and immunochemical reactivities of carboxy-modified human serum albumin.

Seven carboxy modified and four amino modified derivatives of human serum albumin have been prepared and studied by optical rotatory dispersion measurements, gel filtration, immunoelectrophoresis, immunodiffusion and by use of an ammonium sulphate technique. It is found that carboxy groups are of major importance for the maintenance of the structure and function of human serum albumin, and that carboxy modification has a much more profound effect than amino modification has to the same relative extent.

Ammonium Sulfate↗
PubMed1989

A review of the application of chiroptical methods to analytical chemistry.

The analytical applications of the two most important chiroptical methods, optical rotatory dispersions (ORD) and circular dichroism (CD), have been surveyed, emphasizing the methods corresponding to the profile of the Journal of Pharmaceutical and Biomedical Analysis. After a brief introduction of the ORD and CD methods, the advantages and drawbacks of the application of the two methods have been described, and compared, and the calibration of ORD and CD instruments have been given. The analytical applications have been divided as follows: the use of ORD and CD in identification studies, the direct determination of optically active substances, the determination of optically active compounds after chromophore group formation, determination of optically inactive substances via their products with optically active compounds. The difference chiroptical methods increasing the selectivity of measurements and the chiroptical titrations have been summarized. Stress has been laid on the applicability of selective chiroptical detectors (based on laser light, too) used in liquid chromatography and the future developments have been mentioned.

Circular Dichroism↗
PubMed1975

Conformational changes in the progesterone binding globulin-progesterone complex.

An improved purification procedure for the progesterone-binding globulin (PBG) of the pregnant guinea pig has been developed utilizing sulfopropyl Sephadex, a strong cation exchanger, in the first step. The method exploits the low pI (2.8) and favorable acid stability of the glycoprotein. Subsequent chromatographies on DEAE-cellulose and Sephadex G-200 afford a highly purified PBG that exhibits the previously observed polydispersity (R.M. Burton et al. (1974), Biochemistry 13, 3554-3561). Circular dichroism, optical rotatory dispersion, and difference uv spectra all indicate the purified protein to undergo a conformational transition upon forming a complex with a steroid ligand. The CD and ORD spectra cannot be interpreted in terms of tertiary structure probably due to carbohydrate contributions. However, the difference spectra indicate strong perturbation of both a tryptophan residue and the steroid chromophore in the complex.

Animals↗
PubMedDate not supplied

alpha and piDL helical states of alternating poly(gamma-benzyl D-L-glutamate) in solution.

As in solid state, strictly alternating poly(gamma-benzyl D-L-glutamate) in solution can adopt two different helical conformations. Besides the alpha helix, a second helical conformation is found at higher temperatures in dioxane and chloroform, the properties of which correspond to that of the piDL4 helix. As the molecules have a finite length a screw sense is favored for both helical forms thus giving rise to optical activity allowing the study of the transconformation by optical rotatory dispersion and circular dichroism besides infrared and dielectric measurements. Thus, as the temperature is raised the equilibria right-left handed alpha helices and alpha-piDL helical forms can be followed. The favored screw senses are determined by the number of interacting side chains for the alpha helix and by the number of hydrogen bonds which are formed in the piDL helical conformation. The side chain-side chain interactions in the alpha helix are experimentally shown to be attractive.

Glutamates↗
PubMed1979

Conformational changes accompanying the binding of antithrombin III to thrombin.

The conformational aspects of the binding of antithrombin III to thrombin were investigated by difference spectroscopy, circular dichroism, and optical rotatory dispersion. The CD and ORD studies indicate an increase of 6--8% in alpha-helix content at the expense of the beta structure, while the results from difference spectroscopy showed an increased exposure of approximately seven tyrosine residues. In the presence of heparin there is a slightly greater increase in helicity which is accompanied by exposure of an average of two tryptophan and one tyrosine residues. These spectral results indicate that the thrombin-antithrombin III complex formed in the presence of heparin differs in its conformation from that produced in its absence.

Antithrombin III↗
PubMed1992

Occurrence of free D-aspartic acid in marine macroalgae.

The biologically rare D-aspartic acid was found in extracts of marine macroalgae. DL-aspartic acid was isolated from the Phaeophyta, Hizikia fusiformis, by ion-exchange chromatography, and crystallized from aqueous ethanol. It was characterized by optical-rotatory-dispersion analysis and reversed-phase HPLC analysis. The crystal consisted of equal amounts of D and L isomers. The presence of free D-aspartic acid was verified in 15 species from among 20 species of marine macroalgae by the same method (HPLC). Generally, Phaeophyta contained a high concentration of D-aspartic acid in contrast with the low concentration in Phycophyta and Rhodophyta. Particularly in Costaria costata, Hizikia fusiformis and Sargassum yezoensis, belonging to Phaeophyta, D-aspartic acid was found in concentrations proportional to those of L-aspartic acid.

Aspartic Acid↗
PubMed1975

Denaturation and proteolytic digestion of porcine low-density lipoprotein in aqueous guanidine hydrochloride solutions.

The denaturation of porcine low-density lipoprotein (LDL) in aqueous guanidine hydrochloride (GuHCl) was studied by flotation velocity experiments, optical rotatory dispersion and fluorescence spectroscopy. The denaturation of LDL occurred between 2 and 4M GuGCl, where small sigmoidal changes in iptical rotation and fluorescence intensity were noted. The hydrated density of the native LDL was 1.036g/cm-3 and this remained constant upon denaturation in 4M GuHCl. The slope of the flotation coefficient-solvent density curve was 35 per cent less for denatured LDL than for the native LDL. Since there is no indication of splitting of LDL in 4M GuHCl, it is natural to interpret the result in terms of an increase of the translational frictional coefficient by 50 per cent. The observed changes in optical rotation, fluorescence intensity and flotation coefficient in 4M GuHCl were readily reversed and native LDL was recovered after removal of GuHCl by dialysis. Proteolytic treatment of denatured LDL produced digested LDL which had a hydrated density of 1.021g/cm-3 corresponding to the loss of 30 per cent of apo-LDL. The digested LDL behaved like a compact, globular particle in aqueous NaCl solution and in 4M GuHCl. These results can best be interpreted by a model of the LDL particle in which approximately 30 per cent of apo-LDL is exposed to the solvent, such that it can be reversibly denatured by GuHCl and at the same time is easily avalable to proteolytic enzymes, whereas the rest of apo-LDL is tightly associated with lipids and possibly buried inside the lipid moiety. SDS-polyacrylamide gel electrophoresis of the digested LDL revealed four major peptide fragments with sizes ranging from 70,000 to 100,000 daltons. We believe that the method and results described in this paper will have meaningful applications in the study of membrane proteins.

Animals↗
PubMedDate not supplied

[Secondary structure of the polyhedral protein of the nuclear polyhedrosis virus of Bombyx mori and some of its fragments].

The secondary structure of polyhedral protein of the nuclear polyhedrosis virus of Bombyx mori and some of its fragments has been investigated by circular dichroism and optical rotatory dispersion. It has been shown that the protein contains 6% alpha-helices and 26% beta-structures at pH 10.5. The conversion of beta-pleated sheets to alpha-helices after the treatment with sodium dodecyl sulfate was observed. A correlation between the number of alpha-helices in the fragment BrCN-V and its ability to aggregate in aqueous solutions was observed. It was suggested that the COOH-terminal region of polypeptide chain of polyhedral protein makes a considerable contribution to the aggregation of subunits of the polyhedral protein.

Animals↗
PubMed1975

Broad-specificity proteinase inhibitors in Scopolia japonica (Solanaceae) cultured cells. Isolation, physicochemical properties, and inhibition kinetics.

Proteinase inhibitors were isolated from Scopolia japonica cultured cells. Isolation procedures involve concentration by a hydrophobic resin of Diaion HP-20, decolorization by Duolite A-7, affinity chromatography on trypsin-Sepharose, and Bio-Gel P-4 chromatography. It was found that the proteinase inhibitors from S. japonica cells are a mixture of at least five components. For the inhibitory components except one, amino acid analyses, measurements of sedimentation equilibrium and optical rotatory dispersion (ORD) were carried out. The inhibitors were shown to be the polypeptides with molecular weights in the range of approximately 4000 to 6000. In addition, one of them was found to have approximately 15% alpha-helical conformation by the Moffitt-Yang analysis of ORD data. The inhibitors were found to have potent inhibitory activity for trypsin, chymotrypsin, plasmin, kallikrein and pepsin but not for papain with synthetic and natural substrates. These inhibitors formed stable complexes with trypsin and chymotrypsin in an equimolar ratio, and their inhibitory mechanisms for both enzymes were of non-competitive type.

Amino Acids↗
PubMed1979

Interaction of human apohemoglobin with inositol hexaphosphate.

Experiments of sedimentation velocity and equilibrium indicate that in the presence of inositol hexaphosphate the degree of polymerization of apohemoglobin is shifted in favor of the formation of tetramers, with a maximum effect when the concentration of the polyphosphate is 1 mM. Above this concentration, a redissociation of the system into dimers is promoted. This phenomenon is probably due to the binding of inositol hexaphosphate to apohemoglobin with a stoichiometry higher than 1 mol of polyphosphate/4 subunits. The optical rotatory dispersion spectrum of apohemoglobin is also modified by its interaction with inositol hexaphosphate suggesting a small increase in the helical content of the protein. Measurements of circular dichroism in the near-UV region of the spectrum indicate that the environment of the aromatic chromophores of the protein such as tyrosine, phenyalanine, and tryptophan is not affected by the interaction. The presence of inositol hexaphosphate decreases the rate of reaction of the beta-93 cysteinyl residues of apohemoglobin with both p-mercuribenzoate and N-ethylmaleimide, suggesting a conformational change of the protein also at the level of its tertiary structure.

Apoproteins↗
PubMed1977

Stereochemical studies of the pyruvate kinase reaction with (Z)- and (E)-phosphoenol-alpha-ketobutyrate.

(Z)-and (E)-phosphoenol-2-ketobutyrate were synthesized. [3-2H]-2-Ketobutyrates were formed from both isomers in the pyruvate kinase reaction in 2H2O and were converted to chiral propionates. Authentic (2S)-[2-2H]propionic acid was also prepared, and the optical rotatory dispersion curves of the propionates were compared. The rotation compared with standard propionate at 240 nm of sodium (2R)-[2-2H]propionate from the Z isomer was 47% (i.e., 53% was RS), and of (2S)-[2-2H]propionate from the E isomer was 29% (i.e., 71% was RS). Protonation at C-3 of the 2 si, 3 re face of the pseudosubstrates would have yielded (2R)- and (2S)-[2-2H]propionates from the Z and E analogues, respectively. An explanation offered for the nonstereoselective protonation that occurred is dissociation of the enol from the enzyme and subsequent random protonation in solution.

Animals↗
PubMed1975

Further studies on the binding of divalent cations to the phosphoglycoprotein phosvitin.

Dialysis equilibrium measurements at 25 degrees indicate that, at pH 6.8 and at a concentration of 1.0 times 10(-10) 3 M MnC12 or CoC12, phosvitin binds 113 Mn2+ and 120 Co2+. The binding is cooperative at low cation concentrations. The number of Mg2+, Ca2+, Mn2+, and Co2+ bound is not affected by temperatures of up to 60 degrees; however, the cooperactivity is enhanced. Optical rotatory dispersion and circular dichroism studies indicate that a conformational change occurs on binding of Mn2+ and Co2+ which parallels the one produced by Ca2+ and reported elsewhere [Grizzuti, K., and Perlmann, G.E. (1973), Biochemistry 12, 4399]. The conformational changes induced by Mg2+ and Mn2+ follow different paths. Upon binding of Mn2+ and Co2+ the intrinsic viscosity, [eta], of phosvitin decreases from about 0.5 to 0.03 dl/g, while Mg2+ and Ca2+ decrease [eta] to 0.048 dl/g. The ultraviolet absorption spectrum of phosvitin is altered upon binding of Ca2+, Mn2+, and Co2+, but not upon binding of Mg2+; an increase of the temperature to 60% has no further effect on the spectra.

Binding Sites↗
PubMed1976

beta Structure of aqueous staphylococcal enterotoxin B by spectropolarimetry and sequence-based conformational predictions.

Conformations of the globular protein staphylococcal enterotoxin B have been examined experimentally by ultraviolet circular dichroism (CD) and visible optical rotatory dispersion (ORD). Chen-Yang-Chau analysis (Chen, Y.-H., Yang, J.T., and Chau, K. H. (1974), Biochemistry 13, 3350) of the far-ultraviolet CD spectrum of native enterotoxin B revealed (assuming an average helix length of 11 residues) 9% alpha helix, 38% beta structure, and 53% random coil. A fourfold increase in alpha-helix was observed for enterotoxin exposed to 0.2% sodium dodecyl sulfate, behavior typical for globular proteins of low helical content. Values of -40 to -50 for the Moffitt-Yang parameter b0 calculated from visible ORD suggested 6-13% alpha helix in native enterotoxin. Application of a new predictive model (Chou, P. Y., and Fasman, G. D. (1974), Biochemistry 13,222) to the amino acid sequence of enterotoxin B indicated 11% alpha helix, 34% beta structure, and 55% coil in native enterotoxin. The excellent agreement for the amount of alpha and beta conformation utilizing different optical and predictive methods indicates beta structure as the dominant secondary structure in native enterotoxin B. Most of the beta structure is predicted by Chou-Fasman analysis to reside in two large regions of antiparallel beta sheet involving residues 81-148 and residues 184-217. Such highly cooperative regions of anti-parallel beta sheet account for the slow unfolding of enterotoxin B in concentrated guanidine hydrochloride and rapid folding of guanidine hydrochloride denatured enterotoxin B to native conformation(s) (Warren, J.R., Spero, L., and Metzger, J. F. (1974), Biochemistry 13, 1678). A more than twofold increase in alpha-helix content with a small diminution in beta structure was detected by CD and ORD upon acidification of aqueous enterotoxin to pH 2.5. Thus, the beta structure of enterotoxin B appears to resist isothermal denaturation and constitutes a stable interior core of structure in the enterotoxin molecule.

Amino Acid Sequence↗
PubMed1975

State and reactivity of tryptophyl residues in two bacterial proteases from Sorangium sp.

The state and reactivity of tryptophyl residues in two proteolytic enzymes from Sorangium sp. were investigated by means of the following methods: spectrophotometric oxidation of tryptophans with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, and H2O2 in dioxane, optical rotatory dispersion, ultraviolet difference spectrophotometry, solvent perturbation and viscosity measurements. Out of two tryptophyl residues/molecule of alpha-lytic protease, one appears to be completely buried, while the other seems to be exposed. None of these two residues seem to be responsible for the activity of the enzyme. The beta-lytic protease undergoes an irreversible conformational transition between pH 5.0 and 3.5. Out of total four tryptophyl residues/molecule, only one is fully exposed at neutral pH. The other three are gradually exposed in the pH transition region. The degree of exposure and the dimensions of "cavities" shielding tryptophyl residues were estimated. The tryptophyl residues of of beta-lytic protease do not seem to participate in substrate binding or the active site; they are rather one of the determinants of the conformational state of the enzyme.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1976

On the allosteric transition between the structures of high and low ligand affinity in carp hemoglobin.

The variation of magneto-optical rotatory dispersion with pH for carp deoxyhemoglobin in the presence and absence of inositol hexaphosphate was interpreted as a pH-induced allosteric transition between the structures of high and low ligand affinity (the R and T states in terms of the two state model of cooperativity). Increasing the pH from 6 to 11 causes a decrease in the fraction of molecules in the T state from 1 to 0.65. In the absence of inositol hexaphosphate the pH dependence of this fraction has a midpoint at 7.8, addition of inositol hexaphosphate shifts this midpoint by 1.5 units toward high pH. From the analysis of the data obtained and the pH dependences of functional properties (Tan, A.L., Noble, R.W. and Gibson, Q.H. (1973) J. Biol. Chem. 248, 2880-2888) the parameters of the two state model of cooperativity for carp hemoglobin were estimated.

Allosteric Site↗
PubMedDate not supplied

[Comparison of conformational possibilities of polypeptides representing the terminal segments of different histones].

Regular polypeptides--models of the N-terminal fragments of histones H2A and H4 and the C-terminal half of histone H1 were synthesized. Conformations of these polypeptides were investigated by using the methods of circular dichroism and optical rotatory dispersion. It was shown that all polypeptides studied in aqueous solutions at neutral pH and at low temperature (+2 degrees C) had the conformation of left-handed helix (LHH) or poly-L-proline type. The neutralization of positive charges of side groups at alkaline pH of screening of charged groups at a high ionic strength (up to 1 M NaF) results in increase of the degree of defectness of this conformation. There occur no transition of LHH to such an ordered conformation as alpha-helix or beta-sheet structure or complete disappearance of LHH. The influence of temperature, 80% ethanol and 1% sodium dodecylsulphate on the structure of these polypeptides was also studied. Differences in conformational potencies of two studied groups of polypeptides which are the models of the terminal fragments of various histones were discovered and associated with different biological functions of these histones in chromatin.

Circular Dichroism↗
PubMed1976

Properties and subunit structure of pig heart pyruvate dehydrogenase.

Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

Amino Acids↗