Circular dichroism studies of ampullosporin-A analogues.

Ampullosporin A (AmpA), a 15mer peptalbol containing seven Aib residues is able to induce pigmentation on Phoma destructiva and hypothermia in mice, as well as to exhibit a neuroleptic effect. A circular dichroism study of ampullosporin A and its analogues was carried out in organic solvents with different polarities and detergent micelles to determine the relationship between their conformational flexibility and biological activities. The analogues were obtained by modifying the N- and C-termini of ampullosporin A. Furthermore, Gln and Leu were systematically substituted by Ala and Aib residues were replaced by Ala and/or Ac6c. To estimate the helicity of the analogues, the CD spectrum of AmpA recorded in acetonitrile was correlated to its crystal structure. All analogues displayed similar CD curve shapes in organic solvents with the ratio between two negative band intensities R = [theta]n-pi*/[theta]pi-pi* < 1. In acetonitrile, most of the analogues adopted a 70%-85% helical structure, which was higher than the average of 40%-60% obtained in TFE. In detergent micelles, the analogues were distinguishable by their CD profiles. For most of the biologically active analogues, the CD spectra in detergent micelles were characterized by a R ratio > 1 and increased helicity compared with those recorded in TFE, suggesting that the interaction of the peptides with the membrane and peptide association was necessary for their hypothermic effect.     

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.     

Circular dichroism studies of sheep beta-lipotropic hormone.

The far ultraviolet circular dichroism spectra of sheep beta-lipotropic hormone (beta-LPH) were recorded under different conditions of pH, temperature, salt concentration, and solvent composition. Results confirm the stability of the hormone in strong basic or acidic solutions; moreover, temperatures up to 50 degrees C do not seem to affect noticeably the conformation of beta-LPH. However, increasing the NaC1 concentration or addition of dioxane in the solution brings about a conformational transition of the chain, interpreted as an increase in the helical content. The method of Yang (Chen, Y.H., Yang, J. T. & Martinez, H. M. (1972) Biochemistry 11, 4120-4131) was used to compute the proportion of helical, beta, and unordered forms of the hormone chain. The proportions are compared with those obtained from Fasman's predictive method (Chou, P. Y & Fasman, G. D. (1974) Biochemistry 13, 211-221 and Chou, P. Y. & Fasman, G. D. (1974) Biochemistry 13, 222-245) based on the known amino acid sequence of beta-LPH.     

Folding crystallization of DNA. Circular dichroism studies.

CD spectra of DNA monocrystals are extremely different from spectra of psi-DNA or DNA-histone H1 or DNA-polylysine complexes. They are discussed to be dependent on the supramolecular organization of DNA in the condensed form, and they are not in contradiction to the previously proposed seven folding model of DNA in chromatin. Conclusions are drawn with regard to the interpretation of chromatin CD spectra.     

Circular dichroism studies of an oligo-alpha-thymidylate and of its interactions with complementary sequences.

An octathymidylate synthesized with the alpha anomer of thymidine has been studied using circular dichroism. Its conformation has been compared to that of its analogue containing the naturally occurring beta anomer. In both compounds some degree of intramolecular stacking is present as indicated by the shapes of the circular dichroism spectra and their variations with temperature. As its beta-analogue the alpha-octathymidylate binds to its complementary sequences containing beta-nucleosides. Only complexes with 1A:1T stoechiometry were observed. Binding to ribose- containing oligomers and polymers is much stronger than binding to deoxyribose-containing analogues. Circular dichroism spectra provided evidence for a difference between the geometry of the various complexes formed with an alpha-oligothymidylate and those formed with its beta-anomer-containing analogue.     

Glycosylated proline-rich peptide of human parotid saliva. Circular dichroism study.

The direct monitoring of sugars such as lactose, maltose, saccharose is not only useful at the applied point of view but also at the fundamental point of view for studying enzymology, especially in microbiology and fermentation. Benzyme systems were extensively used in solution for analytical applications in industry and medicine. The progress in the field of immobilization of bienzyme systems [1-3], especially within membranes [4-5], makes possible the production of new analytical devices. From the studies dealing with concentration profiles in artificial enzyme membranes [14], evidence was obtained for a well defined relationship between the local concentration of a metabolite and concentration of the first substrate in the bulk solution. In the described systems a substrate is transformed into glucose within a membrane, the glucose is then transformed in gluconic acid with a local oxygen consumption. The local pO2 level is linked to the glucose oxidase velocity, which is only linked to the glucose production, that is to say to the concentration of the first substrate. The enzyme electrode is based on the transformation of kinetic phenomena (reaction rates) into absolute values (local concentrations) through the diffusion-reaction coupling process. The manufacture of magnetic enzyme electrodes [6] allows convenient use of the active sensors. The pO2 electrode has some adventages, namely the specificity based on the selectivity of the gas permeable membrane and the linear relationship between the oxygen and the output of the electrode. pCO2, pH, ion electrodes give a logarithmic response as a function of the concentration. The grafting of a multienzyme system on a sensor allows a study of sequential systems in a defined context with a measurement of the local concentration of the metabolites. The tool is useful for both kinetics [4] and regulation studies [5].     

Circular dichroism studies of some arginine-vasopressin analogues

CD spectra of arginine-vasopressin (AVP) and of its analogues substituted in position 1 and/or 7 were measured in aqueous solution at different pH values. The shapes of the CD spectra of AVP analogues substituted in position 1 are strongly influenced by the type of group attached to the beta-carbon of residue 1. The substitution of the proline residues in position 7 by N-methylalanine also leads to a change in conformation of the peptide. The differences in the CD spectra are interpreted in terms of conformational changes, which are due to the interaction of the tyrosine side chain with neighbouring residues (for 1-substituted analogues of AVP), or to that between the hexapeptide ring and acyclic tripeptide chain (for 7-substituted analogues).     

Conformational characteristics of luliberin. Circular dichroism and fluorescence studies.

By circular dichroism and fluorescence spectroscopy, the conformation of luliberin (luteinizing hormone-releasing hormone) has been investigated under various conditions of pH and solvents. Several structural parameters have been defined which seem predominant for the maintenance of the hormone in some privileged conformation(s). Formation of an intramolecular hydrogen bond between CO (His) and NH (Ser) seems likely when dissolving the hormone in organic solvent such as dioxane. Energy transfer has been demonstrated between Tyr and Trp residues. Calculation of the energy-transfer efficiency at different pH's allowed us to estimate in the range of 10 A the distance which separates these residues. Evidence is also provided for a charge-transfer interaction between protonated histidine and tryptophan. These data suggest that, when luliberin has organized structure (under appropriate surrounding conditions), its conformational pattern would resemble that of beta-turn structure in which a beta bend would exist at the level of the aromatic residues.     

NADPH-cytochrome P-450 reductase. Circular dichroism and physical studies.

NADPH-cytochrome P-450 reductase was purified from hepatic microsomes of phenobarbital and hydrocortisone-treated rats by detergent solubilization and column chromatography. This membrane protein contains 31 mol per cent hydrophobic amino acid residues, 6 half-cystine residues, and a single tryptophan residue as determined by amino acid analysis after mineral or organic acid hydrolysis. The free mobility of cytochrome P-450 reductase in sodium dodecyl sulfate was identical to that of several soluble proteins used as standards (i.e. ovalbumin, bovin serum albumin, erythrocuprein, beta-galactosidase). Molecular weight estimates from sedimentation equilibrium studies in the presence of guanidine hydrochloride (76,500) are consistent with those determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at various per cent gel concentrations (79,000 to 80,000). Computer analysis of circular dichroism spectra of cytochrome P-450 reductase in the far ultraviolet region indicated the presence of 34 per cent alpha helical and 16 per cent beta structure. The amount of random structure was calculated to be 50 per cent.     

Circular dichroism studies on human chorionic somatomammotropin

The conformation of human chorionic somatomammotropin has been studied by means of circular dichroism spectra. The protein appears to contain about 45% α-helix in the native state. Circular dichroism bands in the region of side chain absorption have been assigned to phenylalanine and tryptophan residues. Tentative assignments has also been made to bands probably arising mostly from tyrosine residues. The stability of the native structure has been assessed by challenging the protein with four perturbing solvents. With the exception of 0.1 n NaOH which produced permanent denaturation, all conformational changes produced by the perturbants were fully reversible. In addition, the monomer molecular weight has been evaluated by gel filtration and osmotic pressure measurements. A value of 21,600 ± 900 was found by osmotic pressure at pH 8.4. The results have been compared with similar findings on human pituitary growth hormone and ovine pituitary lactogenic hormone.     

Circular dichroism studies of cobalt substituted lentil lectin

A recent method has been developed to effect metal ion substitution at the Mn2+ site in the lentil lectin (Bhattacharyya et al. (1984) Biochem. Biophys. Res. Commun. 124, 857-862). We report here the preparation of cobalt substituted lentil lectin, containing Co2+ at the S1 site and Ca2+ at the S2 site. The cobalt derivative possesses full saccharide binding activity and can be used for spectroscopic studies. The near UV and visible CD spectra of the derivative are shown, and its spectral properties are compared with various cobalt complexes of concanavalin A.