Properties of some 3-nitrotyrosyl elapid venom cardiotoxins

Nitration of the invariant Tyr-22 in Hemachatus haemachates cardiotoxin 12B did not greatly decrease lethality, and the haemolytic potency towards guinea-pig erythrocytes remained unchanged. This residue is thus non-essential for cardiotoxin to exert its biological action. Nitration of Naja haje annulifera and Naja melanoleuca cardiotoxins VII1 decreased but did not abolish the lethalities and haemolytic potencies. Thus Tyr-25 and Tyr-51 were concluded to have no direct functional role in cardiotoxin lethality. The pKa values of the phenolic hydroxyl groups of the tyrosine residues appeared to be important for certain properties of cardiotoxin in solution. No evidence could be produced to show that Tyr-51 is unreactive to nitration under normal (non-denaturing) conditions.     

Reversed-phase and hydrophobic-interaction high-performance liquid chromatography of elapid cardiotoxins

The separation of proteins by hydrophobic-interaction HPLC and reversed-phase HPLC depends upon differences in the hydrophobicity of accessible surface groups. The elution order of a group of snake venom cardiotoxins was found to vary between these two HPLC methods. Circular dichroism spectroscopy showed that the eluant acetonitrile-trifluoroacetic acid used for reversed-phase HPLC altered the conformation of the toxins, whereas the salt-buffer eluting medium used for hydrophobic-interaction HPLC did not affect toxin conformation. The retention times of cardiotoxins on reversed-phase HPLC are therefore influenced by their conformational instability in the eluting medium which causes partial or complete unfolding. Hydrophobic interaction is clearly the preferred method with which to correlate the "surface hydrophobicity" of cardiotoxins and their biological effects.     

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 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 apolipoprotein E in model complexes and cholesterol-rich lipoproteins: lipid contribution

Lipid-free apolipoprotein E (apo E) and canine apo E HDLc, a cholesterol-rich lipoprotein containing apo E as the only apolipoprotein, show very different circular dichroism (CD) spectra. To determine the cause of the spectral difference, we estimated the CD contribution of phospholipid, cholesterol, and cholesteryl ester in liposomes and microemulsions. We prepared microemulsions, containing nearly equal amounts of egg phosphatidylcholine (PC) and cholesteryl oleate (mean diameter 320 A), by an injection technique. Both microemulsions and cholesterol-containing liposomes exhibit intense negative CD bands in the far-ultraviolet region. Lipids contribute about 20% of the spectral difference between apo E and apo E HDLc at 222 nm, and about 60% of the spectral difference at 208 nm. The remainder of the spectral difference is attributable to lipid-protein interaction corresponding to a 15-30% increase in helicity of apo E. CD analysis indicates that the helical content of apo E in apo E HDLc resembles that in the ternary complex apo E-PC-cholesterol (or apo E-PC-cholesteryl ester) more than that in the binary complex apo E-PC, suggesting that cholesterol affects the conformation of apo E. Our data indicate that in going from a lipid-free state to a lipid environment, apo E undergoes a random to helix transition, assuming the maximal helicity predicted from its primary structure.     

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 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.     

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.     

Theoretical pi-pi absorption, circular dichroic, and linear dichroic spectra of collagen triple helices

Absorption, CD, and LD spectra of the π-π* transition near 200 nm are calculated for poly(Gly-X-Y) (X,Y = Gly, Ala, Pro) in four conformations proposed for collagen like triple helices in the recent literature. A dipole interaction model is used with the same optical paramenters as in previous studies of polypeptide spectra. The CD spectra are sensitive to backbone structure and amino acid composition, although the experimentally observed negative peak near 200 nm is a general feature of most the calculated spectra. Interchain interactions significantly affect the CD spectra in most cases. Calculations for (Gly-Pro-Ala)₃ and (Gly-Ala-Pro)₃ in the triple helical structure of Fraser, MacRae, and Suzuki [(1979) J. Mol. Biol. 129 , 463–481] show absorption, CD, and LD spectra in fairly good agreement with experiment. The characteristics of the π-π* normal modes responsible for the calculated spectra are compared with those of the component bands resolved from the experimental spectra of collagen by Mandel and Holzwarth [(1973) Biopolymers 12 , 655–674].     

Circular dichroic properties and average dimensions of DNA-containing reverse micellar aggregates

With the aim of investigating the compartmentation of nucleic acids and surfactant aggregates, we have studied the circular dichroic properties of DNA solubilized in reverse micelles. DNA incorporated in AOT/isooctane reverse micelles (AOT=bis-2-ethyl-hexyl sodium sulfosuccinate) assumes an anomalous circular dichroism (CD) spectrum with the characteristic features of a psi spectrum. Older literature observations could therefore be confirmed that attribute these spectral changes to the fact that the reverse micelles induce the formation of a condensed form of DNA. A dynamic light scattering (DLS) characterization of the DNA-containing micellar solutions was carried out, and three populations of aggregates in a polar solvent are observed, with an average radius centered at 5, 100 and 1000 nm, respectively, all three containing DNA. Several forms of DNA, including a plasmid, have been investigated. The formation of 1 microm-large aggregates depends on the DNA concentration and such aggregates disappear in the course of a few hours. Conversely, the 100 nm aggregates are stable for at least 1 day and contain DNA in a normal spectral state at low concentration and in a condensed form-it is the characteristic psi spectrum-in a higher concentration range. The solubilization of DNA in reverse micelles brings about unexpected larger structures in hydrocarbon solution, and whereas the very large component can be with all likelihood be attributed to clusters of smaller reverse micelles, the components at 100 nm radius appear to be a quite stable and characteristic feature of DNA-containing reverse micelles.     

On the analysis of circular dichroic spectra of proteins.

A new method is presented for analyzing circular dichroism spectra. The method employs integrals over the data and calculates the alpha-helical, beta-sheet, and random coil content of the proteins from such integrals. It is shown that the analyzed alpha-helical content is usually reliable to within 5%, beta-sheet values are somewhat less reliable, and random coil values are least reliable. Curve fitting techniques are shown to be misleading. The method has a number of advantages over existing procedures.