Change In Optical Rotation Research Articles

Concentration Dependence of the Order‐Disorder Transition of Carrageenans

The temperature-induced changes in optical rotation which are associated with the order-disorder transition of carrageenans have been analysed to investigate the concentration dependence and hence to attempt to derive the number of polysaccharide chains involved in the ordered species at equilibrium. The consequences of the known polydispersity have been analysed by computer-modelling and it is shown that this need not cause misleading results. For iota-carrageenan segments of 0.1 M NaCl and 0.1 M KCl solution the results point to a two-chain state and therefore support the proposed double helix model. For chi-carrageenan segments the apparent average number of molecules involved in the ordered state at equilibrium is 3.7, suggesting that if double helices form here they aggregate further. The method of investigation may be generally applicable to measuring the number of chains which associate in ordered states in aqueous solutions of polysaccharides.

Physical Characterization of Amylose‐Fatty Acid Complexes in Starch Granules and in Solution

AbstractPrevious differential scanning calorimetry studies of native starch have shown an endotherm at ∼ 100°C on heating, which is attributed to melting of an amylose‐lipid complex. In the present work we have shown that this transition is fully reversible (ΔH ≈ 0.4 ± 0.1 cal g−1 on heating and cooling) and displays substantial thermal hysteresis (Tm≈76°C on cooling). On removal of lipid by extraction with methanol the transition is lost, but re‐appears on addition of sodium palmitate to de‐fatted granules. Closely similar thermal behaviour, including hysteresis, is observed on addition of palmitate to amylose. Further evidence of complex formation between amylose and palmitate is the almost total disappearance of high resolution 1H n.m.r. signal, indicating loss of conformational mobility by the polysaccharide chain. Formation of the complex is also accompanied by a large reduction in optical activity. Using an established correlation between D‐line optical rotation and the dihedral angles between adjacent residues we have compared the observed optical rotation with values calculated for specific ordered structures of amylose. The measured optical rotation values indicate adoption in solution of a novel right‐handed V‐amylose structure (calculated [α] D=104°; found = 100±5°) rather than the expected left‐handed form characterized in the solid state (calculated [α] D=157°). Analysis of optical rotatory dispersion curves shows that in each case the changes in optical rotation on complex formation arise predominantly from changes in the intensity of the known polysaccharide backbone transition at 161 nm.

Radiation Chemical Studies of Protein Reactions: Dependence of Restorative Amino Acids on Radiation Dose

Abstract Changes of specific rotation of protein irradiated with Co-60 gamma-rays were restored by some amino acids, such as mono-sodium 1-glutamate. Loss of this recovery effect in some amino acids by irradiation was studied by polarimetry and infrared spectometry. Optical rotation of protein-amino acid solutions increased with an increasing radiation dose given to the amino acids. Changes in optical rotation with irradiation may be due to the loss of the recovery effect of amino acids for the irradiated protein molecule. Changes in infrared spectra increased with an increasing radiation dose given to the amino acids. Such changes in infrared spectra with irradiation may be due to destruction of some radicals in the amino acid molecule.

Spectropolarimetric Back-Titration Method for the Determination of Milligram Amounts of Platinum (IV)

Abstract Spectropolarimetric titrimetry was first introduced by Kirschner et al.1,2 and has enjoyed considerable recent success. This was due principally to the introduction of several relatively inexpensive photoelectric polarimeters and subsequent improvements in their design and versatility3,4. Fundamental to this technique is the use of a photoelectric polarimeter to monitor the change in optical rotation of the solution as the titration proceeds. Parameters must be selected so that an inflection in the optical rotation occurs in the vicinity of the equivalence point. Spectropolarimetric titrimetry has been applied to acid-base titrations, as well as to different metals systems2. The applications of this technique have been significantly broadened by Pearson et al., who utilized the two chiral and stereospecific amino-polycarboxylic acids (R)-(-)-1,2-propylenediaminetetra-acetic acid, R-(-)PDTA, and (R, R)-(-)-trans-1,2-cyclohexanediaminetetraacetic acid, R, R(-)CDTA, in the volumetric titrimetric ana...

Kinetics of Thermal Denaturation of β-Lactoglobulin at pH 2.5

Thermal denaturation of β-lactoglobulin at pH 2.5 was studied at temperatures ranging from 60 to 130C. Changes were followed by measurements of specific optical rotation [α]58925 and solubility at pH 4.5. Denaturation, as determined by both methods, was irreversible and was evident only at 75C or above, a higher temperature than 65C reported for denaturation at neutral pH. Also, the cooperative transition temperatures were higher (83 to 84C) at pH 2.5 than at pH 6.7 (68 to 69C).Changes in specific rotation indicated that at all temperatures above 75C denaturation proceeded in two stages. The first stage (up to 5min) was faster than the subsequent stage (5 to 60min). Specific rotations of the pH 4.5-insoluble fraction resolubilized at pH 2.5 and the pH 4.5-soluble fraction, readjusted to pH 2.5, increased steadily with duration and temperature of heat treatment, indicating progressively increased unfolding of protein molecules. Stable [α]58925 were not reached after heating for 60min. The final [α]58925 was much lower than that (∼100) achieved in the presence of concentrated urea or guanidine hydrochloride. Changes in optical rotation could not be fitted into a simple kinetic scheme.The extent of denaturation measured by loss of solubility at pH 4.5 indicated it followed pseudo first-order or two consecutive first-order reactions. Denaturation rate constants were determined from the slope of the lines. The Arrhenius plot of the logarithm of the apparent denaturation rate constant as a function of the reciprocal of the temperature (°K) enabled calculation of the activation energy of denaturation (∼43 Kcal/mol) for β-lactoglobulin heated at pH 2.5. The kinetics of thermal denaturation at pH 2.5 have been compared with those at neutral pH.

The measurement of mean temperatures in plant and soil studies by the sucrose inversion method

A polarimetric procedure is described for determining exponential mean temperatures (θe) from the rate of inversion of sucrose solutions buffered at pH values between 1 and 3. An equation is given for calculating the pH values appropriate for various temperature ranges and time scales. The procedure used for monitoring air and soil temperatures over a 5 year period at a network of sites in Aberdeenshire was to expose 5 ml volumes of sucrose solution at pH 2.06 in sealed polythene tubes. Each month the changes in optical rotation were measured with a sensitive automatic polarimeter and fresh tubes exposed. The values of θe were 2°C higher than arithmetic means obtained from standard thermometric devices. Observations at elevations between 275 and 686 m a.s.l. demonstrate the lapse of temperature with altitude and illustrate the usefulness of θe measurements.

A physico-chemical study of heparin: Evidence for a calcium-induced co-operative conformational transition

The conformations of heparin in aqueous solution in the presence of sodium, potassium, magnesium and calcium cations were studied using circular dichroism, optical rotation, nuclear magnetic resonance and equilibrium dialysis. Potassium and magnesium cations, when added to sodium heparinate solutions, cause small chiroptical changes. Binding of calcium ions gives rise to large changes in both optical rotation and circular dichroism. This is indicative of a major change in chain conformation, which is also manifest in 13C and 1H n.m.r. § § Abbreviations used: n.m.r., nuclear magnetic resonance; p.p.m., parts per million. Equilibrium dialysis suggests one mole of calcium bound per mole of tetrasaccharide, which n.m.r. indicates to be appropriately sulphated iduronateglucosamine-iduronate-glucosamine. The calcium is chelated by two iduronate carboxyl groups. Proton-proton coupling constants, determined by convolution difference spectroscopy and Carr-Purcell sequences, indicate that, over the temperature range 285 to 353 K, the iduronate ring is best described as 1C 4( l) and the glucosamine residue as 4C 1( d) for both sodium and calcium forms. The conformational change induced by calcium is ascribed to rotation around the glycosidic linkages. The binding process is co-operative and the binding constant of 10 3 to 10 4 m −1 is biologically significant. The findings are consistent with intramolecular binding. Hence, this study represents the first report of a polysaccharide undergoing a cation-induced intramolecular disorder-order process. The authors postulate that a function of the post-polymerization epimerization of d-glucuronate to l-iduronate is the attainment of the precise geometry required for co-operative calcium binding with consequent modulation of the flexibility of the tetrasaccharide units.

The mucilage of Opuntia ficus-indica

The mucilage extracted from the cladodes (modified stems) of Opuntia ficus-indica contains residues of d-galactose, d-xylose, l-arabinose, l-rhamnose, and d-galacturonic acid. Seasonal variation in the sugar composition of the mucilage has been investigated. Fractionation studies indicate that the mucilage is essentially homogeneous. The rate of release of the constituent sugars and the change in optical rotation on mild hydrolysis coupled with the results of chromic acid oxidation suggest that the mucilage contains α-arabinofuranosyl, β-xylopyranosyl, β-rhamnopyranosyl, β-galactopyranosyl, and α-galactopyranosyluronic acid residues. The results also suggest a core containing galacturonic acid, rhamnose, and galactose, to which xylose and arabinose are attached in peripheral positions.

The Pfeiffer effect, outer-sphere complexation, and the absolute configuration of dissymmetric coordination compounds

Abstract

Pressure jump method with detection of optical rotation and circular dichroism

A pressure jump technique for fast kinetics using optical rotation and circular dichroism detection is presented and a suitable cell design for both detection methods is discussed. The detectability of changes in optical rotation far away from the Cotton region is considered, and measurements made on a pressure-dependent folding reaction of the protein serum albumin are given as an application.

Complex Gels of Proteins and Acidic Polysaccharides. Part II. The Effect of Electrostatic Interaction on Structure Formation in Complex Gels of Gelatin and Alginate

AbstractComplex gelatin‐alginate gels prepared from insoluble (type I) and soluble (type II) complexes are studied by the thermomechanical technique and by polarimetry. Gels of both types are thermoirreversible at concentrations higher than 6% and at weight fractions of alginate of 0.3 or higher. Suppression of complexation between gelatin and alginate by introduction of a neutral salt or by increasing pH above pIG leads to formation of thermoreversible gels the thermomechanical properties of which are similar to those of gelatin gels. Change of optical rotation on heating is less for complex gels than for gelatin gels of the same concentration.

The nature of alkylurea and urea denaturation of α-chymotrypsinogen

The optical rotatory dispersion of α-chymotrypsinogen in aqueous solution became less levorotatory upon the addition of ethyl-, propyl-, or butylurea; less negative values for the Moffitt-Yang parameter, α 0, were also obtained. This change in optical rotation of α-chymotrypsinogen induced by the alkylureas was similar in direction and magnitude to that observed for alcohols but was opposite to that caused by unsubstituted urea. It appears, therefore, that the alkylureas share with the alcohols an ability to rearrange α-chymotrypsinogen into a non-native yet regularly ordered conformation. The effectiveness of the alkylureas and alcohols as denaturants for this protein increased in the order ethyl<propyl<butyl derivatives. An identical rank-order was observed for the ability of the alkylureas and alcohols to diminish attractive forces between aliphatic groups, as measured by a model system based upon the extent of aggregation of glass beads coated with methyl groups. These findings indicate that the denaturing action of alkylureas for α-chymotrypsinogen is a function of the substituted aliphatic group and is predominantly hydrophobic in character. Non-hydrophobic interactions of unsubstituted urea with α-chymotrypsinogen appear to be critical for unfolding of the protein to a random-coil configuration.

Temperature stability of phospholipase A activity—I Bee (Apis mellifera) venom phospholipase A 2

Enzymic activity of bee venom phospholipase A 2 has been studied at high temperatures using dispersed egg yolk and sonicated dipalmitoyl and dicaproyl lecithins as substrates. With egg yolk substrate the maximum activity was observed at 65°C and both dipalmitoyl and dicaproyl lecithins gave a maximum at 60°C. At 60°C the enzyme showed loss of activity in the presence of 0·7 M urea. The loss of activity at ca. 60°C could be due to the physical change in the nature of the substrates and/or due to the denaturation of enzyme at this temperature. Preincubation of enzyme at temperatures up to 90°C for 45 min gave the same temperature optimum of 65°C with egg yolk substrate which shows that the heat denaturation, if it occurs, is reversible. O.R.D. studies of pure bee venom phospholipase A 2 showed no change in optical rotation from 10 to 90°C. In spite of the constant optical rotatory dispersion of the protein up to 90°C, sudden loss of enzyme activity was noticed above 60°C with the lecithin substrates.

Apparatus for rapid measurement of optical rotation changes

An apparatus for rapid measurement of optical rotation changes has been developed in the face of large absorption changes with time resolutions of 10 μsec (sensitivity 0.018°) and 4 msec (sensitivity 0.0016°). Applications are briefly described for the study of rapid transient processes of reduction reaction of cytochrome c with dithionite with the stopped-flow technique. The method can be combined with stopped-flow, temperature-jump, and flash photolysis and allows direct observation of rapid conformation events of biomolecules.

The pH Jump Study of Enzyme Proteins

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.

Polarimetric stopped-flow apparatus

A stopped-flow apparatus is described for the measurement of millidegree changes in optical rotation and absorbance changes at wavelengths between 230 and 800 nm. The design utilizes Faraday modulation at 2.5 kHz and signal processing by selective filtering and phase sensitive detection. Observed rms noise levels are reported for the wavelength range 230–450 nm: representative values for nonabsorbing solutions are 0.2 mdeg at 450 nm (15 nm bandpass, 9 msec time constant) and 3 mdeg at 260 nm (8 nm bandpass, 9 msec time constant). It is shown that these figures correspond to the theoretical shot-noise limited performance of the instrument. The use of the apparatus is exemplified by studies of the hydroxide ion-catalyzed mutarotation of glucose, and the displacement of NADH by NAD+ from the NADH-liver alcohol dehydrogenase complex.

Denaturation and Proteolytic Digestion of Porcine Low-Density Lipoprotein in Aqueous Guanidine Hydrochloride Solutions1

The denaturation of porcine low-density lipoprotein (LDL) in aqueous guanidine hydrochloride (GuHCl) was studied by flotation velocity experiments, optical rotatory dispersion and fluorescence spectroscopy. The denaturation of LDL occurred between 2 and 4M GuGCl, where small sigmoidal changes in iptical rotation and fluorescence intensity were noted. The hydrated density of the native LDL was 1.036g/cm-3 and this remained constant upon denaturation in 4M GuHCl. The slope of the flotation coefficient-solvent density curve was 35 per cent less for denatured LDL than for the native LDL. Since there is no indication of splitting of LDL in 4M GuHCl, it is natural to interpret the result in terms of an increase of the translational frictional coefficient by 50 per cent. The observed changes in optical rotation, fluorescence intensity and flotation coefficient in 4M GuHCl were readily reversed and native LDL was recovered after removal of GuHCl by dialysis. Proteolytic treatment of denatured LDL produced digested LDL which had a hydrated density of 1.021g/cm-3 corresponding to the loss of 30 per cent of apo-LDL. The digested LDL behaved like a compact, globular particle in aqueous NaCl solution and in 4M GuHCl. These results can best be interpreted by a model of the LDL particle in which approximately 30 per cent of apo-LDL is exposed to the solvent, such that it can be reversibly denatured by GuHCl and at the same time is easily avalable to proteolytic enzymes, whereas the rest of apo-LDL is tightly associated with lipids and possibly buried inside the lipid moiety. SDS-polyacrylamide gel electrophoresis of the digested LDL revealed four major peptide fragments with sizes ranging from 70,000 to 100,000 daltons. We believe that the method and results described in this paper will have meaningful applications in the study of membrane proteins.

An Optical Rotation Temperature Jump Apparatus for Helix-Coil Transition of Biopolymers

ABSTRACT A temperature jump apparatus to observe optical rotation changes and thus investigate the conformational change of biopolymers has been proposed. Heating is achieved with an electrical pulse using a coaxial cable as the capacitor, and temperature rise time is shorter than 0.5 μsec The maximum sensitivity of the apparatus is 0.001deg; of rotation per cm of oscilloscope screen. Relaxation associated with the helix-coil transition of poly-α, L-glutamlc acid has been observed. The pH and degree of polymerization dependencies of the relaxation time have also seen examined.

AN OPTICAL ROTATION STOPPED FLOW APPARATUS

Abstract A stopped flow apparatus has been proposed for optical rotation changes. This apparatus, even when the signals of optical rotation changes are masked by absorption changes, can detect only the signals of optical rotation changes. The apparatus is examined by several model reactions: The maximum sensitivity is 0.01 degree of rotation, the dead time ca. 80 msec. The 50% change of transmittance does not affect the signals of optical rotation changes.

Conformational induction in copolypeptides of the form AnBm.

AbstractThe polymerization kinetics and optical rotatory properties of AnBm polypeptides have been studied, where A = D,L‐Tyr, D,L‐TyrZ, D,L‐LysZ, L‐AspOBzl, or L‐Asp‐ONBzl, and B = D,L‐GluOR (R = Me or Bzl). In most cases where An and Bm prefer the same helix sense, the polymerization of A N‐carboxyanhydride (initiated by Bm in dioxane) shows first order kinetics and produces a monotonic change in optical rotation, while if opposite helix senses are preferred, the kinetics are multiphasic and the change in rotation reverses direction after the addition of several residues. The rotation change in the latter case is interpreted to mean that the helix in the A block is initially induced to take the nonpreferred sense, as originally suggested by Doty and Lundberg from similar observations on (D‐GluOBzl)n‐(L‐GluOBzl)m. It is found here that the CD spectra for the latter polymer show the sign changes required by this hypothesis. The optical rotation curves and CD spectra for (D,L‐Tyr)n‐(L‐GluOBzl)20 suggest, by analogy, that (L‐Tyr)n prefers the same helix sense as (L‐GluOBzl)n. However, it is found that the opposite conclusion is equally consistent with the data if one considers the effects of possible changes in side‐chain conformation on these data in accordance with the calculated CD spectra of Chen and Woody. The optical rotation curves for (D‐GluOBzl)n‐(L‐GluOBzl)20, (D‐Tyr)n‐(L‐GluOBzl)20, and (L‐Tyr)n‐(L‐GluOBzl)20 are all found to be consistent with a two‐state equilibrium model in which the A block initially takes on an induced conformation and has an increasing tendency to revert to its preferred conformation as n increases. It is concluded that in both D‐Tyr and L‐Tyr the side‐chain and/or the backbone conformation is induced by the neighboring L‐GluOBzl block, and the data do not distinguish which type of change is occurring. These results are discussed in connection with other observations bearing on the helix sense of (L‐Tyr)n.