Optical Rotatory Dispersion Parameters Research Articles

Buoyant titration of bovine mercaptalbumin chemically modified at the carboxyl groups

Bovine mercaptalbumin has been modified at the carboxyl groups by means of 1-ethyl-3 (3-dimethylisopropyl) carbodiimide and glycine amide. A stable derivative was obtained after modification of 54 groups. The modified protein differed only slightly from the unmodified one with respect to the optical rotatory dispersion parameters ao, bo and χc. This preparation was studied in the analytical ultracentrifuge in a CsCl gradient in the pH-interval from 2 to 11. The buoyant densities were determined and compared with those of the unmodified protein in the same pH-interval. The contribution from the carboxyl groups to the buoyant density is discussed and compared with results obtained with other proteins and synthetic polypeptides.

Conformational Studies on Modified Proteins and Peptides: IV. CONFORMATION OF LYSOZYME DERIVATIVES MODIFIED AT TYROSINE OR AT TRYPTOPHAN RESIDUES

Abstract Conformational investigations have been carried out on derivatives of lysozyme in which tyrosines 20 and 23 were nitrated (NT2-lysozyme), or in which the nitrotyrosine residues had been reduced to aminotyrosine (AT2-lysozyme). Also, a derivative modified at the 6 tryptophan residues by reaction with 2-nitrophenyl sulfenyl chloride (NPS6-lysozyme) was studied. In optical rotatory dispersion measurements, NT2-lysozyme and AT2-lysozyme showed equal rotations at the negative 233-nm minima and at the positive 199-nm maxima. Also, they had identical b0 values. These two derivatives were more rotatory than lysozyme. On the other hand, NPS6-lysozyme showed an appreciable degree of unfolding evidenced by a decrease of all its optical rotatory dispersion parameters. Measurements of the reduced molar ellipticities showed that the present three derivatives, like lysozyme, give a negative circular dichroism band at 208 nm and a shoulder around 220 nm. Results were in agreement with optical rotation measurements. The rotatory behavior of lysozyme and the derivatives showed no change in the pH range 7 to 3. The conformational changes revealed by optical rotatory dispersion and circular dichroism measurements were also shown by increase in disulfide reducibility. Both NT2-lysozyme and AT2-lysozyme exhibited appreciable disulfide reducibility (one bond) relative to lysozyme (0.03 bond), and the great unfolding in NPS6-lysozyme was also confirmed by the large increase in accessibility of its disulfide bonds (2½ bonds). Changes in conformation obtained on binding of each of these proteins with sodium dodecyl sulfate were monitored by disulfide accessibility. It was shown that NT2-lysozyme and AT2lysozyme assume ‘relaxed’ conformation with identical disulfide accessibility (two bonds). The conformation of lysozyme also became ‘relaxed’, although to a lesser extent than the tyrosyl derivatives, on binding with sodium dodecyl sulfate. In contrast to these, NPS6-lysozyme, assumed a ‘constrained’ conformation on binding with sodium dodecyl sulfate. It was concluded that conformational changes take place upon modification of the tyrosine or tryptophan residues and that NT2-lysozyme and AT2-lysozyme had identical conformations. From these results and the previously reported immunochemical behaviors of these derivatives, it may be concluded that one (or both) of tyrosines 20 and 23 is located in an antigenic reactive site in lysozyme. The results also indicated that, although the antigenic reactivity of native proteins is highly influenced by changes in conformation, not every conformational change will exert an effect on the antigenic reactivity. This should be dependent on the protein and the nature of the conformational change.

Physicochemical studies on the aggregation of bovine cardiac tropomyosin with ionic strength.

The aggregation of bovine cardiac tropomyosin as a function of ionic strength has been studied by the techniques of sedimentation velocity, sedimentation equilibrium, osmometry, viscometry, and optical rotatory dispersion. The measurements indicate that in aqueous buffers at neutral pH and at ionic strengths below 0.6, cardiac tropomyosin is heterogeneous and consists of a monomer in equilibrium with its aggregates. Dissociation of the aggregates occurs on dilution to yield a molecular weight species of approximately 70 000, in very good agreement with the value obtained at high ionic strength. These observations essentially parallel similar findings noted for the polymerization of skeletal tropomyosin, with the exception that the cardiac protein shows no tendency to polymerize at high ionic strength. The virtual constancy of all the optical rotatory dispersion parameters with polymerization suggests that the association is probably linear rather than lateral, with no accompanying changes in secondary and tertiary structures of the individual monomers.

Beef Heart Malic Dehydrogenases: VII. Reactivity Of Sulfhydryl Groups And Conformation Of The Supernatant Enzyme

Abstract Only 3 of the 6 sulfhydryl groups of native bovine heart supernatant malate dehydrogenase (S-MDH) react with p-mercuribenzoate (PMB) with no loss of enzymatic activity. In addition, prolonged incubation of S-MDH in 2.6 m urea solutions does not significantly alter the catalytic properties of the enzyme. In 2.6 m urea, however, all 6 sulfhydryl groups of the enzyme react with PMB, with attendant losses in catalytic activity. Under these conditions the rate of inactivation, although considerably slower than the rate of mercaptide formation, closely parallels the rate of change in the optical rotatory dispersion properties of the enzyme. Native S-MDH exhibits an optical rotatory dispersion curve with a distinct shoulder in the 280 to 290 mµ spectral region and a trough at 233 mµ. Changes in these properties and other optical rotatory dispersion parameters were used to index conformational changes of S-MDH accompanying the various treatments. Native S-MDH and fully active S-MDH · (PMB)3 exhibit identical optical rotatory dispersion curves. S-MDH in 2.6 m urea also exhibits an optical rotatory dispersion curve similar to that of the native enzyme. S-MDH · (PMB)3 and S-MDH · (PMB)6 in 2.6 m urea, however, have significantly different optical rotatory dispersion properties, including a disappearance of the 280 mµ shoulder, decreases in [m']233 values, and large decreases in -b0, a0, and λc values. Since the rate of change in these optical rotatory dispersion properties closely parallelled the rate of loss of catalytic activity but not the rate of mercaptide formation, the results were interpreted as indicating that the loss in activity is related to the changes in protein conformation rather than to the actual blocking of sulfhydryl groups.

Nuclear magnetic resonance and optical spectroscopic studies of poly l and poly d alanine

Nuclear magnetic resonance spectroscopy is finding increasing application to studies of conformational changes in biological macromolecules and their synthetic analogues. It has been suggested that the resonance peaks due to backbone protons in helical polymers will be broadened by non-averaging of dipole-dipole interactions and that the areas of these resonance peaks will be a measure of the random coil content of the system. In agreement with others, we do not find this to be the case for poly l and poly d alanine in trifluoracetic acid (TFA)-deuterochloroform (CDCl 3) solvent systems where the optical rotatory dispersion parameters indicate a helix content in excess of 50 per cent while the n.m.r. spectra for these solutions are still fully developed. It is suggested that the peak narrowing process operating in this system is rapid exchange between random coil and helical segments. Others suggest that, although the b 0 values for the TFA-CDCl 3 solvent systems indicate 54 per cent helix content, this o.r.d. parameter is an unreliable guide to helix content and that the polypeptide is helical; furthermore that they are observing for the first time a well developed n.m.r. spectrum for a polypeptide ‘purported to be in a helical conformation’. These conclusions are not supported by the results obtained from studies of poly l and poly d alanine in dichloracetic acid (DCA)-TFA and DCA-CDCl 3 solvent systems. In these systems it is possible to obtain appreciably higher b 0 values (up to 458°) than was observed for the TFA-CDCl 3 solvent system and these increases in b 0 are accompanied by marked broadening and loss of area of the proton resonance peaks. It is suggested therefore that when the helix content is greater than 50 per cent the motions of helical segments become too slow for the peak narrowing process of exchange between helical and random coil forms to be effective. These observations support the earlier suggestions that in fully helical macromolecules the backbone protons resonance peaks will be broadened by non-averaging of dipole-dipole interactions. It is possible to correlate the shifts in the poly l alanine resonance peaks with helix content and it has been suggested that the shifts are due to exchange of the protons between two chemical environments, i.e. the helical and random coil forms. Infra-red studies show, however, that there is a specific interaction between the carboxylic acid molecule and the helical form of poly alanine and we suggest that part of the shifts observed in the resonance peaks may result from magnetic anisotropy effects of the carboxyolic acid molecules complexed with hydrogen bonded amide groups in the helical form. In agreement with others it is suggested that the mode of interaction of the carboxylic acid molecules with poly alanine is a strong hydrogen bonding interaction and not protonation as has been suggested.

Spectroscopic studies of the conformations of histones and protamine

Earlier optical rotatory dispersion studies on calf thymus histone fractions showed that increasing the salt molarity of aqueous solution caused an increase in the optical rotatory dispersion parameters b o and R′ 233 and it was assumed that this effect was due to a partial coil → helix transition in the histone fractions. It is now known that it is not possible, on the basis of low values of optical rotatory dispersion parameters, to decide whether the solutions contain simple mixtures of helix and random coil forms or whether other conformations such as extended β-forms are present, and further studies of these systems have been made using other spectroscopic techniques. Infrared studies of the histone solutions show the presence of a large proportion of β-structures in the solutions and solid films of the lysine-rich fraction F1 while the other fractions are shown to contain mixtures of helix and random coil forms. The formation of extended chain conformation appears to be a property of the lysine-rich fractions and this is related to its possible structural role in chromatin. The increase in the optical rotatory dispersion parameters with increasing ionic strength suggests that the partial coil to helix transition is caused by a reduction in the electrostatic repulsion forces between like-charged sidechains. The conformational changes as a function of pH have been investigated and show that, for all the fractions, increasing the pH also causes a partial coil to helix transition, indicating that most helical segments result from a reduction in repulsion energy between basic charged residues; in the case of the arginine-rich fraction F3, however, it was found that decreasing the pH also caused a partial coil to helix transition and for this fraction it is suggested that some helical segments result from the neutralization of charged acidic residues. From nuclear magnetic resonance spectroscopic studies of these solutions it is inferred that other residues involved in the helical regions of the arginine-rich fractions are the apolar residues alanine, leucine, isoleucine and valien. Since it is known that these fractions are the most tightly bound to DNA and that most of the enhanced stability of DNA in nucleoprotein against thermal deuteration is also due to these fractions, it is suggested that a source of stabilization of the partial nucleoprotein formed between DNA and the arginine-rich histone, additional to the ionic interactions, is hydrophobic interactions between the apolar residues in the helical segments and the lower sections of the deep groove of the DNA molecule.

Optical Rotatory Dispersion of the β-Form of Synthetic Polypeptides in Solution

Optical rotatory dispersion measurements were made on poly-S-methyl- L cysteine (PSMLC), poly-S-carbobenzoxy- L -cysteine (PSCBLC), poly-S-carbo-methoxyethyl- L -cysteine (PSCMELC) and poly-S-carbobenzoxyethyl- L -cysteine (PSCBELC) in chloroform-dichloroacetic acid (DCA) mixed solvent. Infrared absorption spectrum measurements were also carried out for PSCBLC, PSCMELC and PSCBELC. From both measurements, these three poly- L -cysteine derivatives were found to be in an intermolecular β-form in the mixed solvent with low DCA content. A large positive a 0 value of +800 and a small b 0 value of nearly zero were obtained as optical rotatory dispersion parameters for the intermolecular β-form of PSCBLC in solution. The transition from the intermolecular β-form to a random coil conformation was observed for PSCBLC, PSCMELC and PSCBELC in solution, in which the large positive a 0 value changes into a large negative one whereas the b 0 value remains almost constant. Discussion is made as to the stability of the i...

Solvent Perturbation and Ultraviolet Optical Rotatory Dispersion Studies of Paramyosin

Abstract The location of the tyrosyl residues (there are no tryptophyl residues) in paramyosin was studied by solvent perturbation difference spectroscopy, with 20% polyhydroxy perturbants of increasing size from ethylene glycol to sucrose. Of the tyrosyl residues, 35% are not perturbed by any of the solvents, and an additional 10 to 15% apparently lie in crevices which are inaccessible to perturbants with diameters greater than 5 A (i.e. glycerol and larger molecules). These latter groups may be involved in the intermolecular aggregation phenomenon. Increasing concentrations of guanidine-HCl increase the magnitude of the denaturation blue shift in four steps. The first two of these steps coincide with the first two steps in the helix-coil transition and account for the exposure of about 68% of the 38 tyrosyl groups to the solvent. The remaining 12 tyrosyls are exposed in two steps during the breakdown of the final third of the helix and are considered to be the same 12 groups inaccessible to all perturbants. The ultraviolet optical rotatory properties of native α-helical paramyosin from 315 to 190 mµ were studied in detail. Statistical analyses of the Moffitt equation showed that it adequately describes the dispersion data in the 315 to 240 mµ region, if λ0 is taken as 220 mµ, until the molecule becomes less than 30% helical. Beyond that point the one-term Drude equation is sufficient. The Moffitt parameters are shown to be colinear with [m']232 for the whole helix-coil transition. By comparison with optical rotatory dispersion parameters for random polypeptides, paramyosin retains approximately 10% order in 7 m guanidine. Since this order cannot be destroyed by heating to 50° or by 8 m guanidine-HCl and the molecule refolds completely upon removal of the guanidine, hydrophobic interactions appear responsible for this order.

The Effects of Solvent Environment on the Optical Rotatory Dispersion Parameters of Polypeptides: I. Studies on Poly-γ-Benzyl-l-Glutamate

The constancy of the Moffitt optical rotatory dispersion parameters for polypeptides in different solvents was tested by dispersion measurements on poly-gamma-benzyl-L-glutamate in fifty-five solvents and solvent mixtures. b(0) was not constant but varied linearly with the refractive index of the solvent according to the equation -b(0) = 1701 - 730.3 n(8). This variation could not be explained by changes in configuration of the polypeptide. a(0) also showed a trend with solvent index but the values were widely scattered. lambda(0) did not show a statistically significant dependence on solvent index. The variation in b(0) can be interpreted as an effect of solvent polarizability on the frequencies of optically active transitions.

Characterization of a Reversibly Formed Enzyme Complex in the Reaction of Chymotrypsin with Diisopropyl Fluorophosphate

Considerable evidence that the interaction of cr-chymotrypsin with substrates or inhibitors results in conformational changes in the enzyme has been reported (l-6). This evidence was provided in part by experiments with covalently bound enzymesubstrate complexes which indicated that the optical rotatory dispersion parameters (l-3) and the thermodynamic parameters of reversible, temperature-induced phase transitions (4) differ from those of the free enzyme and depend on the nature of the substrate. Other evidence was provided by measurements of difference spectra between enzyme and covalently bound enzymesubstrate complexes (5, 7). The difference spectrum (see, for example, Fig. 3b), with a major peak at 290 rnp, was observed to be the same whether the group attached to the enzyme was diisopropylphosphoryl, acetyl, trimethylacetyl, or cinnamoyl. Recent data on x-ray diffraction from crystals of native y-chymotrypsin and of a covalently bonded inhibitor-enzyme complex also suggest that some deformation of the protein structure accompanies the formation of the enzyme-inhibitor complex (8). In order to understand the interrelationships of the steps which are associated with the spectral changes, the changes in specific rotation of the enzyme, and the catalytic reaction, we have undertaken a kinetic study of the reaction of cY-chymotrypsin with diisopropyl fluorophosphate in the pH range of 2.0 to 10.0 (9). This reaction yields covalcntly bonded diisopropylphosphoryl-oc-chymotrypsin and HF in stoichiometric amounts (10). In this paper we are reporting equilibrium and kinetic studies of this reaction at pH 2.0, including data on the spectral changes, the change in optical rotation, and the phosphorylation and inhibition of the enzyme. We are also reporting on the chymotrypsin-catalyzed hydrolysis at pH 2.0 of a specific substrate, N-acetyl-n-phenylalanine ethyl ester, and on the spectral changes of the enzyme which accompany this reaction.