Circular dichroic properties of rat liver arginase

• 1.1. Rat liver arginase contains over 50% of the α-helical and about 10% of β-pleated structures.
• 2.2. The manganese ions do not cause the changes in the far ultraviolet CD spectra of the enzyme, whereas they induce the optical activity at 280 nm.
• 3.3. The circular dichroic changes at near ultraviolet region coincide with the activation of arginase.

     

Circular dichroic properties and conformation of thionicotinamide dinucleotides.

The spectroscopic properties of the 3-thioamide analogues of coenzymes NAD and NADH (sNAD and sNADH) have been investigated in order to obtain information about their conformational properties. In particular, ultraviolet absorption and circular dichroism properties of solutions in phosphate buffer pH 7 and ethanol were studied. Also equimolar mixtures of AMP and sNMN(H), obtained by cleaving the coenzymes with phosphodiesterase, were investigated using the same solvents. The appearance of a couplet around 260 nm, which is not present for the mixture of sNMN and AMP, suggests a stacking interaction of the two aromatic moieties in sNAD. This conclusion is further substantiated by a hyperchroism of the ultraviolet absorption band in the 260-nm region in both sNAD and sNADH. The comparison of the ultraviolet and circular dichroic properties of intact and cleaved coenzymes in the different solvent systems makes it possible to single out the bands which are more sensitive to conformation changes (i.e. to open-stacking equilibrium) and those which are sensitive to solvent effects only.     

Circular dichroic spectroscopy of non-human alpha-macroglobulins

Bovine, chicken and frog alpha-macroglobulins and ovomacroglobulin were studied by circular dichroic spectroscopy over the region 205-250 nm. All four spectra exhibited negative ellipticity with minima at about 215 nm similar to that reported for human alpha 2-macroglobulin. On reaction of the alpha-macroglobulins with trypsin, the spectrum of each of the four changed similarly. However, these proteins exhibited different conformational changes when treated with methylamine. These differences were exploited to determine which characteristics of alpha-macroglobulins correlate with changes in circular dichroic spectroscopy.     

Circular dichroic spectra of elapid cardiotoxins

Cardiotoxins isolated from elapid snake venoms constitute a chemically homogeneous family of molecules. Within this group several biologically different subclasses exist. We report a comparative analysis of the structure of 20 cardiotoxins using circular dichroism, immunological methods and secondary-structure prediction. It is shown that cardiotoxins fall within two structural subclasses. Toxins of group I are characterized by (a) CD spectra having an intense positive band close to 192.5 nm and a negative trough at 225 nm with no positive band around 230 nm, (b) strong cross-reactivity with a polyclonal antiserum specific for Naja nigricollis toxin gamma and (c) a high tendency to form a reverse turn in the region of position 11. Toxins of group II are characterized by (a) CD spectra displaying a much weaker positive band at 192.5 nm, a negative band around 210 nm and a positive band at 230 nm, (b) little cross-reactivity with the aforementioned antiserum and (c) a high reverse-turn potential at position 31. It is suggested that the observed differences result from differing curvatures in the antiparallel beta sheet which constitutes the main secondary structure of cardiotoxins.     

Circular dichroic properties and conformation of thionicotinamide dinucleotides bound to horse-liver alcohol dehydrogenase.

The interaction between horse liver alcohol dehydrogenase and the oxidized and reduced forms of the 3-thionicotinamide--adenine dinucleotide coenzyme analogues (sNAD and sNADH) has been investigated by ultraviolet absorption, fluorescence and circular dichroism. The fluorescence of sNADH is enhanced when bound to the enzyme, and the protein fluorescence is quenched by both sNADH (60--65%) and sNAD (65%). The possible origin of the larger quenching produced by sNAD with respect to that of NAD is discussed. Coenzyme dissociation constants have been determined by monitoring the quenching of the protein fluorescence, and some kinetic consequences of these dissociation constants are discussed. The dichroic properties of various enzyme complexes have been investigated, and are discussed in terms of conformational changes and environmental changes during coenzyme binding. The conformation of sNAD bound to the enzyme in the presence of trifluorethanol and the conformation of sNADH bound to the enzyme in the presence of isobutyramide have been analyzed in particular detail. Also the circular dichroic spectrum of the apoenzyme is discussed in terms of contributions of the aromatic amino acid residues in the highly resolved 240--310-nm region and in terms of helix content in the 220-nm region.     

Circular dichroic properties of the tyrosine residues in tetrazole analogues of opioid peptides.

CD studies on tetrazole analogues of opioid peptides show that peptides sharing the same N-terminal sequence, H-TyrPsi[CN(4)]Gly-, give very large Cotton effects of the Tyr side chain in the near-UV region. CD spectra of five such peptides: H-TyrPsi[CN(4)]Gly-Gly-Phe-Leu-OH (I), H-TyrPsi[CN(4)]Gly-Phe-Pro-Gly-Pro-Ile-NH(2) (II), H-TyrPsi[CN(4)]Gly-Phe-Pro-NH(2) (III), H-TyrPsi[CN(4)]Gly-Phe-Gly-Tyr-Pro-Ser-NH(2) (IV), and H-TyrPsi[CN(4)]Gly-Phe-Asp-Val-Val-Gly-NH(2) (V), and two others for comparison: H-Tyr-GlyPsi[CN(4)]Gly-Phe-Leu-OH (VI) and H-TyrPsi[CN(4)]Ala-Phe-Gly-Tyr-Pro-Ser-NH(2) (VII), were measured in methanol, 2,2,2-trifluoroethanol, and water at different pH values. The spectra show that the conformations of the Tyr(1) residue in peptides I-V are very similar in all solvents used but differ distinctly from those observed for VI and VII. Strong Tyr bands in the aromatic region result probably from the rigid structure of the common N-terminal part of peptides I-V. These bands are weaker for IV, which maybe due to the presence of a second Tyr residue in that peptide, giving an opposite contribution to the CD spectrum as that arising from Tyr1. It seems that the rigid structure of the N-terminal part of I-V results from the interaction of the Tyr(1) side chain and the tetrazole ring. The CD bands of the Tyr residues of VI and VII are much smaller than those of I-V in all solvents, except VII in trifluoroethanol (TFE) where Tyr bands comparable in intensity to those of I-V are observed. This spectral property may derive from the same sign contribution of both Tyr residues of VII to the CD spectrum.     

Circular dichroic titration of dihydrofolate reductase with TPNH

Dihydrofolate reductase from Streptococcus faecium shows a marked aromatic side chain Cotton effect in the 260–310 nm region of its circular dichroic spectrum. This effect consists of three distinct ellipticity bands with maxima centered at 305 nm, 295 nm and 270 nm. Titration of the enzyme with TPNH to a 1:1 stoichiometry results in the generation of an extrinsic Cotton effect at ca. 340 nm and a decrease in the magnitude of the side chain Cotton effect. This is the first such example of a TPNH-generated extrinsic Cotton effect. The data suggest the involvement of tryptophyl residues in coenzyme binding.     

Circular dichroic study of conformational changes in ovalbumin

By simulation of the circular dichroic spectra (Greenfield and Fasman (1969)) and using reference spectra of Chen et al. (1974), native ovalbumin was estimated to contain 33% -helix, 5% -structure, and 62% random coil. Ovalbumin resisted conformational changes in solutions of urea and of SDS. However, guanidine induced transition, starting at about 2 M and completing at about 4.5 M. At concentrations exceeding 4.5 M guanidine, ovalbumin existed as 6–7% -helical, 12–13% -structure, and 80–81% random coil. Ovalbumin after denaturation in 6 M guanidine or in 8 M urea (incubated at 4°C for 24 hr) did not recover the native conformation but acquired a new conformation in each case, with a somewhat destabilized helical structure.Abbreviation used CD circular dichroism - SDS sodium dodecyl sulfate     

Circular dichroic spectrum of poly-L-tyrosine in aqueous solution

The UV and CD spectra of poly-L-tyrosine were investigated at pH 10.6 and pH 11.2. At pH 10.6 (μ=0.1), the CD spectrum exhibits a medium positive band at 230mμ, an extremely small negative band at 217mμ, and a large positive band at 200mμ. At pH 11.2 (μ=0.1), a new positive CD band appears at 277mμ while the bands at 230mμ and 217mμ are shifted to longer wavelengths by 15 and 10mμ respectively. These results, together with UV spectral data and a specific rotation- pH profile, suggest that at pH 10.6, poly-L-tyrosine exists in the helical conformation with only a small fraction of its side chains ionized; at pH 11.2, the polypeptide retains its helical structure but with a considerable increase in ionization.     

Activity and stability of catalase in nonionic micellar and reverse micellar systems

Catalase activity and stability in the presence of simple micelles of Brij 35 and entrapped in reverse micelles of Brij 30 have been studied. The enzyme retains full activity in aqueous micellar solution of Brij 35. Catalase exhibits "superactivity" in reverse micelles composed of 0.1 M Brij 30 in dodecane, n-heptane or isooctane, and significantly lowers the activity in decaline. The incorporation of catalase into Brij 30 reverse micelles enhances its stability at 50 degrees C. However, the stability of catalase incubated at 37 degrees C in micellar and reverse micellar solutions is lower than that in homogeneous aqueous solution.     

Yeast glyoxalase I. Circular dichroic spectra and pH effects

Large scale isolation and physicochemical characterisation of yeast glyoxalase I showed that this enzyme contained small amounts of carbohydrates. Circular dichroic spectra of the enzyme measured in the presence and absence of S-(p-bromobenzyl)glutathione indicated perturbation of a tyrosine on binding of this competitive inhibitor. Values of Ki for competitive inhibitors were pH invariant over the accessible pH range.     

Circular dichroic spectroscopy of membrane haemoproteins. The molecular determinants of the dichroic properties of the b cytochromes in various ubiquinol:cytochrome c reductases

The circular dichroism (CD) of dihaem cytochrome b from mitochondrial and bacterial ubiquinol:cytochrome-c reductase (bc1 complex) has been characterized. The dichroic properties of the yeast purified cyt b are very similar to those of the native cyt b within the mitochondrial bc1 complex. The CD spectra in the Soret region of the native cytochrome b present in all species studied show an intense bisignate Cotton effect having a zero-crossing wavelength close to the absorbance maximum. In preparations partially or completely depleted of the low-potential b haem (b1) the CD spectra exhibit a single positive Cotton effect resembling the corresponding absorption spectrum. This is particularly evident in the purified cytochrome b-562 from Rhodobacter sphaeroides R26, which contains only the high-potential b haem (bh). These spectral features together with the reconstitution of the cytochrome b1 haem have been used to resolve the CD contribution of each haem to the CD spectra of cytochrome b. The mechanisms which might be responsible for the optical activity have been examined. It appears that the CD spectra of cytochrome b derive from both the mutual interaction of its two haems (giving rise to exciton coupling) and to the interaction of each haem with nearby aromatic residues, other than the pairs of histidines which coordinate the iron. The dipole coupling between haem and aromatic residues appears to be more important than exciton coupling in the CD spectra of oxidized b cytochromes and correlations have been made between the CD features and the proposed structure of cytochrome b.