Optical rotatory dispersion secondary

Circular dicliroism has been a useful servant to tire biophysical chemist since it allows tire non-invasive detennination of secondary stmcture (a-helices and P-sheets) in dissolved biopolymers. Due to tire dissymmetry of tliese stmctures (containing chiral centres) tliey are biaxial and show circular birefringence. Circular dicliroism is tlie Kramers-Kronig transfonnation of tlie resulting optical rotatory dispersion. The spectral window useful for distinguishing between a-helices and so on lies in tlie region 200-250 nm and hence is masked by certain salts. The metliod as usually applied is only semi-quantitative, since tlie measured optical rotations also depend on tlie exact amino acid sequence. 

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

The physical and chemical properties of the AChR have been elucidated. Optical rotatory dispersion measurements indicate that the receptor consists of about 34% helix and 28-30% P-sheet structure—a high proportion of ordered secondary structure. Some carbohydrates are part of the molecule. The DNA encoding the receptor has been cloned and sequenced, revealing the complete amino acid sequence of the subunits. 

At pH 12, the disulfide and noncovalent bonds are both broken, and the monomer with a sedimentation constant of 1.45 Svedberg units is released. From frictional ratios, the monomer appears to exist as a coil with a diameter of 16 A and a length of 150 A. Analysis of the primary structure of K-casein (Loucheux-Lefebvre et al. 1978) suggests considerable secondary structure in the monomer. 23% a-helix, 31% /3-sheets, and 24% 0-turns. In contrast, other investigators, using several different approaches, obtained a-helix contents ranging from 0 to 20.8% (Bloomfield and Mead 1975). Circular dichroism spectra on the monomer indicated 14 and 31% for a-helix and / -sheet, respectively (Loucheux-Lefebvre et al 1978). An earlier study of the optical rotatory dispersion of the K-casein monomer yielded values for the a-helix content ranging from 2 to 16% (Herskovits 1966). 

The secondary and tertiary structure of a partially purified 7S globulin was examined by Fukushima (7) based on optical rotatory dispersion, infrared and ultraviolet difference spectra. Antiparallel (5 -structure (352) and random coil (60%) predominated with only 5% helical structure present. The contribution of the three structures was calculated from molecular ellipticity values obtained by circular dichroism (11) and from the Moffitt parameters in ORD (11, 12). Between 210 and 250 nm, the experimental CD curve for the 7S protein was similar to the CD curve computed from ORD Moffitt parameters with the major dissimilarity occurring at 208-213 nm. 

Relative to secondary structure, viscosity, sedimentation velocity, ultraviolet difference spectra and optical rotatory dispersion studies (4,24,25) showed that glutenin appears to be an assymetric molecule with a low a-helix content (10-15%). Glutenin contained more a-helix structure in hydrochloric acid solutions and less in urea solutions. The amount of a-helix structure is also influenced by changes in ionic strength (26). 

The trithiocarbonates may prove useful as intermediates for the synthesis of sugar dithiols from epoxides. Ring opening by reductive cleavage with lithium aluminum hydride gives excellent results with aliphatic and ahcyclic trithiocarbonates. When both carbon atoms are secondary, the product is a iraws-dithiol for example, cyclohexene oxide, which is converted into a irans-trithiocarbonate, gives, on reduction, cyclo-hexane-1,2-dithiol. The reaction has been used in the cyclitol series for the preparation of 1,2-dithio-neo-inositol and 1,2-dithio-ir-inositol, from 1,2-anhydro-alZo-inositol. The inositol trithiocarbonates show pronounced Cotton effects in their optical rotatory-dispersion spectra. 

A correlation between content of hydrophobic amino acids and surface activity of five different food proteins partially hydrolyzed with 0.1% pepsin has been reported (58), but exceptions were noted. Protein hydrolysates exhibiting large surface absorption were correlated with large foam stability and a large external hydrophobic region. It was concluded that protein hydrolysates with large surface hydrophobic regions adsorbed more readily at interfaces and rates of surface desorption were lower. However, secondary structures, as measured by optical rotatory dispersion and infrared spectra, and the content of the total hydrophobic amino acids in the protein hydrolysates showed no correlation with their foam stabilities (58). 

Optical activity of natural products may depend on chemical factors such as asymmetric carbon atoms, restricted rotation, etc. These may be termed primary structural features. There are also secondary structures, e.g., helices or random coils, that may confer chirality to a natural product. Optical rotatory dispersion (ORD, i.e., rotation of plane-polarized radiation over a range of wave-lengths usually from approximately 200 to approximately 500/im) has been used in studies of the conformations of many different molecules, including polymers, proteins, and polypeptides [90]. 

Several factors must be considered for a particular biomacromolecular structure application that will affect the choice of spectroscopic methods. These include structural resolution necessary, chemical nature of biomacromolecule (protein, nucleic add, or glycan), amount/concentration of biopolymer available, sample preparation (solid or solution), solvents of interest, and desired structure information (secondary or tertiary structure). Structural resolution varies considerably for the various spectroscopic methods, with X-ray diffraction and NMR providing atomic resolution (high resolution) and ultraviolet (UV) absorption revealing merely information about the polarity of the chromophore s environment (low resolution). X-ray studies require crystals while NMR experiments prefer solutions in deuterated solvent. Solvent preferences can affect the choice of spectroscopic method as, for example, infrared (IR) encoimters strong interference from water, while optical rotatory dispersion (ORD) and circniar dichroism (CD) do not. Some of the commonly used spectroscopic methods in structural analyses of biomacromolecules will be discussed. 

The cobalt(III) trien base hydrolysis inversion needs further elucidation with complete optical rotatory dispersion or circular dichroism curves, in order to determine whether the secondary nitrogen about which the rotation takes place during the oc to P inversion is also inverted. The synthetic method of preparing P-cis isomers from a-cfs isomers... 

Fig. IX-5. Schematic models of DNA-clupeine complexes based on the thermal denatura-tion, optical rotatory dispersion, gel-filtration, and electron microscope studies. Primary complex clupeine molecules wind around the small grooves on the DNA double helix. Secondary complex the primary complexes are joined together in places by protamine bridges. Network a network of nucleoprotamine complexes as can be seen in the electron micrograph (cf. Fig. IX-4) (from Inoue and Ando, 1969)...

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