Ultraviolet Difference Spectra Research Articles

Bovine serum albumin in lithium chloride solutions. Changes in conformation.

The physical state of bovine serum albumin in LiCl solutions at the pH near isoelectric point and room temperature has been studied with intrinsic viscosity, optical rotatory dispersion, circular dichroism and ultraviolet absorption difference spectra. The results indicate that the effects of Li+ take place in two processes. 1. At the concentration of LiCl below 4.0 M, only the general electrostatic effect occurs. There is no conformational change in the protein. 2. At the concentration of LiCl above 4.0 M, a specific effect of the Li+ occurs in which both the secondary and tertiary structures of the protein change. In addition, the protein molecules aggregate. In correspondence with the two processes, two different possible mechanisms are discussed.

Conformational changes and association of chemically modified chymotrypsins

The low pH conformational transitions of a series of modified chymotrypsins have been examined and compared to the association properties of these same proteins in order to determine if the effect of the modification was directly upon the association or indirectly through conformational changes. The modifications of α-chymotrypsin that have been studied are serine-195 modified with acetyl or with phenylmethane sulfonyl groups, His-57 modified by tosylamidophenylethyl chloromethyl ketone or with a methyl group, and Met-192 modified with a nitrophenacyl group. Chymotrypsinogen and native and modified delta-chymotrypsin have also been studied. Ultraviolet difference spectra and optical rotatory dispersion measurements show that the various proteins may be divided into three groups depending upon their response in the low pH transition. These groupings based upon conformational transitions are not divided in the same manner as were those based upon the effect of the modifications on the association process itself. The latter process had earlier been found to correlate with the size of the modification in the active site. From these findings it has been concluded that the effect of the modifications on the association was directly upon the interacting subunit interface and that the observed conformational change is only incidental to this effect. Therefore, the amino acids involved in the association in solution are the same as those involved in the dimerization in the crystal, namely, the active site region of Ser-195, His-57, and Met-192. This is the most direct demonstration that the mode of association of a protein in solution is the same as the mode of association in the crystal.

Role of Tyrosine Residues in Interactions between Casein Components

It was indicated from ultraviolet difference spectra and ultracentrifugal experiments that associations occurred between two casein components (αs- and κ-caseins, β- and κ-caseins and αs- and β-caseins) at lower CaCl2 concentrations (2~3 mm) and that aromatic amino acid residues participated in the associations. Chemical modification studies with 2-hydroxy-5-nitrobenzylbromide indicated that tryptophane residues of each casein component were not essential for these associations. It was also demonstrated by nitration of tyrosine residues with tetranitromethane that tyrosine residues of κ-casein were essential for αs·κ-association and for β·κ-association and that tyrosine residues of αs-casein were important to αs·β-association.Interactions between casein components were also studied at higher CaCl2 concentration (10 mm) which is enough for micelle formation. It was found that tyrosine residues of κ- casein played an important role for the stabilization of αs- and β-caseins. Properties of the nitrated-β-cas...

Porcine Intestinal Calcium-binding Protein: MOLECULAR PROPERTIES AND THE EFFECT OF BINDING CALCIUM IONS

Abstract A calcium-binding protein from porcine intestinal mucosa has been characterized by a variety of physical and chemical techniques. Chromatography on Sephadex G-75 suggests a molecular weight around 12,000, while electrophoresis in sodium dodecyl sulfate-urea-polyacrylamide gel indicates a molecular weight in the region of 7000. Sedimentation equilibrium analyses give evidence for aggregation, with the monomeric form having a molecular weight between 7000 and 9000. The amino acid composition is as follows: Lys11–12, Arg1, Asp7, Thr1, Ser6–7, Glu17, Gly4, Ala5, Val3, Ile3, Leu10, Tyr1, Phe5. There is no histidine, half-cystine, methionine, or tryptophan. The composition requires a minimal molecular weight of 9000. No free terminal amino group was detectable, but digestion with penicillocarboxypeptidases S1 and S2 indicated the following COOH-terminal sequence: .... (Gly, Thr, Asp)Ala-Ile-Val(Phe, Ser)Leu-Lys-GlnOH. The circular dichroism spectrum of the binding protein in the near ultraviolet shows a positive peak at 276 nm with a shoulder at 282 nm characteristic of tyrosine, two positive peaks at 264 nm and 257 nm, and two negative peaks at 268 and 261 nm characteristic of phenylalanine. In the far ultraviolet range two negative bands at 222 nm and 207 nm are observed. Removal of calcium ions causes a marked decrease in the tyrosine band at 276 nm to less than 25% of its initial value, but no difference is observed in the far ultraviolet range. Ultraviolet difference spectra between the calcium-containing and the calcium-free protein show peaks characteristic of tyrosine. No difference in sedimentation rates was observed. It appears that the binding of calcium causes a local perturbance of the environment of the single tyrosine residue, but no gross conformational changes.

Studies on the protein comformation of phytochrome.

Abstract— The extinction coefficients for large rye phytochrome were found to be: Fluorescence and circular dichroism spectra of large‐ and small‐molecular‐weight rye phytochrome give no evidence for a protein conformational change on phototransformation of phytochrome. The large molecule has a fluorescence emission peak at 331 nm when excited at 290 nm, and an excitation peak for this emission at 288 nm. The circular dichroism spectra indicate that large rye phytochrome has about 17–20% a‐helix content, 30%β‐structure and 50% random coil, and that the small rye phytochrome has about 10–13%α‐helix content. The ultraviolet difference spectra for large and small rye phytochrome are similar and differ from the difference spectrum of the small oat phytochrome in the relative size of the 296–298 nm peak. The difference spectra may reflect changes in chromophore absorbance and in the environment of amino acid residues near the chromophore, particularly of tyrosine, and perhaps of tryptophan and cysteine.

The conformational state of methionine residues in the temperature-controlled transition of chymotrypsinogen and -chymotrypsin.

The reaction of the two methionine residues in bovine α-chymotrypsin and chymotrypsinogen A with hydrogen peroxide at pH 3.0 and temperatures varying from 4° to 60° was investigated. Reaction rate constants were determined and compared with those of free methionine. Methionine-192 in α-chymotrypsin has the same reactivity as free methionine over the whole temperature range. This is compatible with the results of X-ray analysis (1) which shows that the sulfur atom is fully exposed. The ratio of the reactivity of methionine-192 in chymotrypsinogen to that of free methionine at temperatures of up to 30° is about 1:40. At higher temperatures this ratio increases markedly. It is concluded that methionine-192 in chymotrypsinogen is partially buried and access to reagents is restricted. No reaction of methionine-180 has been detected up to 25° (α-chymotrypsin) and 30° (chymotrypsinogen), respectively. At higher temperatures there is a steep increase in the reactivity. These results have been interpreted in terms of a two-state transition between a form in which methionine-180 is buried and one in which it is exposed. The calculation of transition temperatures gave a value of 55° for α-chymotrypsin and 60° for chymotrypsinogen. These have been compared with values of 48–51° for α-chymotrypsin and 54–56° for the zymogen obtained from ultraviolet difference spectra and from optical rotatory dispersion studies (2–4).

A kinetic study of the energy storing enzyme-product complex in the hydrolysis of ATP by heavy meromyosin

The hydrolysis of ATP by heavy meromyosin was studied by means of the measurement of the development of enthalpy. The results were compared with the rate of change in the intensity of the ultraviolet difference spectrum of heavy meromyosin. It is shown that as far as the enthalpy change is concerned: (1) most of the excess energy associated with ATP does not directly dissipate into the solution during the rapid hydrolysis of ATP in the initial stage of the reaction but is stored in stable form in an enzyme-product complex, (2) the ultraviolet difference spectrum of heavy meromyosin is due specifically to the reaction via the enzyme-product complex, suggesting that a local conformation of heavy meromyosin is changed because of the excess energy stored in the complex, and (3) the complex dominantly exists during the steady splitting of ATP.

L-Asparaginase from Erwinia carotovora: PHYSICOCHEMICAL PROPERTIES OF THE NATIVE AND SUCCINYLATED ENZYME

Abstract The physicochemical properties of the antileukemic l-asparaginase from Erwinia carotovora have been studied and compared with those of the corresponding clinically effective enzyme from Escherichia coli B. Although both enzymes are tetramers with similar molecular weight, the amino acid compositions are distinctly different and the Erwinia enzyme is more basic as judged by polyacrylamide electrophoresis. Of the 48 to 50 tyrosyl residues in native Erwinia asparaginase, only about 10% ionize with a normal pK, while 30% of these residues in the E. coli enzyme titrate normally. The native tetramer (s20,w = 7.2) is considerably more stable than the corresponding tetramer from E. coli. Asparaginase from Erwinia is only partially dissociated in 8 m urea, whereas the E. coli enzyme is completely converted to the 1.7 S monomer under the same conditions. Guanidinium chloride at concentrations of 3.5 m completely dissociates Erwinia asparaginase. Dissociation is accompanied by the appearance of an ultraviolet difference spectrum with a maximum at 288 nm. The rate of dissociation is markedly increased by the addition of alcohols to the denaturant. Dissociation in the presence of alcohols occurs in two distinct steps whose rates increase as the length of the alkyl side chain of the alcohol increases. Succinylation of the lysyl residues results in marked increases in mobility of the modified enzyme on polyacrylamide gels, but does not affect the state of aggregation of the tetramer or its catalytic activity. In addition succinylation does not affect either the rate of dissociation in guanidinium chloride or the extent of reconstitution to the active enzyme. The presence of five bands on polyacrylamide gel electrophoresis of hybrids prepared from the native and succinylated enzyme indicates that Erwinia carotovora asparaginase is composed of four identical subunits.

Non-linear relationships between oxygen saturation and magnitude of fine structure of ultraviolet oxy versus deoxy difference spectrum in human hemoglobin

Ultraviolet difference spectra of fully oxygenated hemoglobin vs. successively deoxygenated or reoxygenated hemoglobin were determined in the absence and presence of organic phosphates. Magnitude of fine structure in the difference spectrum around 290 nm, which is considered to be a partial reflection of oxygenation-induced changes in quaternary conformation of hemoglobin, was not linearly related to fractional oxygen saturation of hemoglobin of the reference cell. The non-linear feature was influenced by the organic phosphates as predicted by the allosteric model of Monod et al. The present study suggests that the ultraviolet oxy vs. deoxy difference spectrum measurements provide a useful way to examine the validity of the model.

The location of tryptophanyl groups in human and bovine carbonic anhydrases: Ultraviolet difference spectra and chemical modification

1. 1. The effects of 20% sucrose, glycerol, ethylene glycol, polyethylene glycol, dimethyl sulfoxide and 86% 2H 2O on the ultraviolet absorption spectra of bovine carbonic anhydrase and the human carbonic anhydrases B and C have been investigated. The results suggest that 3 ± 1 out of 7 tryptophan residues are accesible to the solvent in the bovine and human C enzymes and 2 ± 0.5 residues out of 6 in the human B enzyme. There is no systematic dependence of the degree of accessibility on the size of the perturbing molecule, but “short range” perturbants consistently give lower values than “long range” perturbants. 2. 2. In the bovine enzyme and the human C enzyme no tryptophan residue appears to have a “normal” reactivity toward 2-hydroxy-5-nitrobenzyl bromide. In the human B enzyme the equivalent of one residue can be modified without significant losses of enzymic activity and native structure. 3. 3. Acid denaturation appears to result in an incomplete exposure of tryptophanyl groups. Addition of urea or guanidine·HCl to the acid-denatured enzyme results in spectral changes suggesting a further unfolding of the molecule. It is estimated that the transfer of all the tryptophanyl groups of the native bovine enzyme to water is associated with Δε 292 nm approximately — 11 000 M −1 · cm −1 or an average value per tryptophan residue approximately −1600 M −1 · cm −1. 4. 4. A survey of a model of the crystal structure of human carbonic anhydrase C has been made. Only one of the tryptophan residues is buried in the interior of the molecule. The remaining 6 groups have surface positions. One of these surface groups would be considerably more exposed to the solvent than the remaining 5 groups.

Conformational Changes of αs Casein by Heating

Conformational changes of αs-casein by heating were investigated by measuring ultraviolet difference spectra. The ultraviolet difference spectra at elevated temperature against 5.5°C were measured in various ionic strengths and pHs. Thermal effects of the difference spectra were cancelled by comparing with the spectra of model compounds such as lysozyme and ribonuclease, and the blue shift of αs-casein spectra was observed at above 30°C in these all experimental conditions. This shift was considered to mean unfolding of the αs-casein molecule. The aggregation of αs-casein was observed above ionic strength of 0.4 by heating. These heat-induced changes were reversible until the aggregation was observed.

Ultraviolet difference spectra of the lactose repressor protein

Ultraviolet difference spectra of the lactose repressor have been studied under various conditions. Changes in the aromatic residues of the protein upon addition of inducers, but not anti-inducers, are found. The extent of exposure of aromatic residues to solvent perturbation is also found to differ in the presence of inducers and anti-inducers. These differences are interpreted in terms of a change in the protein structure at points distinct from the ligand binding site.

The binding of α-ketoglutarate in a binary complex and in a ternary complex with NADP + by l-glutamate dehydrogenase

l-Glutamate dehydrogenase ( l-glutamate: NAD(P) + oxidoreductase (deaminating EC 1.4.1.3)), cooperatively binds NADP + and α-ketoglutarate in a highly stable dead-end complex exhibiting a near ultraviolet difference spectrum characteristic of red shifts of tryptophan and oxidized nicotinamide absorbance. The requirements of an intact amide on the micotinamide moiety and two carboxyl groups on the substrate for a 200-fold heterotropic cooperativity in binding are demonstrated by the use of coenzyme and substrate analogs. The agreement of the binary complex dissociation constants calculated from the concentration dependence of the formation of the abortive complex with those determined directly shows that both coenzyme and α-ketoglutarate are bound at the same site in the binary and ternary complexes. By analogy it is inferred that NADP + and l-glutamate bind to the enzyme to form the active complex with a cooperativity similar to that demonstrated for the dead-end complex. The ability of the enzyme to form the NADP + -α-ketoglutarate dead-end complex and other stable complexes is pertinent to the catalytic mechanisms proposed for glutamate dehydrogenase. This ability also provides a mechanism through which both the in vivo direction and rate of catalysis can be selectively and sensitively controlled by the cooperative binding of the reactants and products themselves.

Tetrahydrobiopterin, a cofactor in mitochondrial electron transfer. A soluble transfer system.

Submitochondrial particles, obtained from sonified rat liver and beef heart mitochondria, exhibit a strong increase of oxygen consumption in the presence of tetrahydropterin. As with to intact mitochondria, ultraviolet difference spectra indicate a concomitant reduction of cytochromes c and a/a3. A soluble electron transfer system, which consists of the essential components tetrahydropterin, cytochrome c, and cytochrome oxidase, is able to reduce molecular oxygen by NADH or NADPH. Submitochondrial particles and mitochondria are activated by cytochrome c in the same way, but less effectively. Radioactivity of 14C‐labelled tetrahydrobiopterin is incorporated into mitochondria in the range of 1/4 to 2/3 of equilibrium distribution. The possible physiological function of tetrahydropterins in the control of cellular reducing power is discussed.

Comparison of acid‐induced conformation changes between 7S and 11S globulin in soybean seeds

AbstractThe acid‐induced conformation changes between 7 and 11 S globulin, the major storage proteins in soybean seeds, were compared by ultraviolet difference spectra, ultracentrifugation and optical rotatory dispersion. Maximum denaturation occurred at approximately pH 2 in both and dissociation of the proteins into subunits and unfolding of the polypeptide chains were observed simultaneously. However, both proteins showed apparent differences in their readiness to undergo acid‐induced denaturation. The differences were particularly remarkable in the presence of 0.1 ionic strength sodium chloride.

Some Effects of Calcium Ions on the Structure of Bovine Pancreatic Deoxyribonuclease A

Abstract The effects of divalent metal ions on the circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of bovine pancreatic deoxyribonuclease have been studied. Both the CD and ORD spectra of DNase undergo significant changes below 250 nm upon the binding of calcium to the enzyme. The maximum increase in ellipticity at the 215 nm minimum in the CD spectrum is 950 deg cm2 per decimole. Fifty per cent of the maximum transition occurs near 10-5 m CaCl2. MnCl2 gives a smaller increase at 215 nm of 290 deg cm2 per decimole while MgCl2 causes no change. Calcium is able to protect DNase completely against inactivation by trypsin. Half-maximum protection is achieved near 1.3 x 10-5 m CaCl2. Although some protection against trypsin inactivation is given by MnCl2, and less by MgCl2, at no concentration is either metal able to protect completely. Calcium-induced changes in the CD spectrum and in susceptibility to trypsin must not involve any large changes in shape or volume of the protein since no change in the sedimentation coefficient could be observed. Concentrations of 10-4 m to 10-2 m CaCl2 cause intensification of the CD bands between 310 nm and 250 nm. The same calcium concentration range induces a large ultraviolet difference spectrum in DNase. This indicates that calcium causes significant perturbations of tryptophan and tyrosine residues in DNase.

Conformational equilibria in a synthetic copolypeptide.

AbstractA synthetic copolymer of L‐glutamic acid and L‐tyrosine (23:1) with molecular weight 17,000 was examined conformationally as a function of pH, using circular dichroism, difference spectrophotometry, fluorescence, potentiometic titration, and high‐resolution nuclear magnetic resonance (220 MHz). A water‐dioxan mixture (2:1) was used to avoid complications due to aggregation (which was shown by infrared spectroscopy to lead to the formation of β‐structures). In the α‐helical form the tyrosine residues generate a sizeable negative Cotton effect in the near ultraviolet; this is a consequence of perturbation of the chromophores by the helix, and not of tyrosine‐tyrosine interactions, which are known to give rise in the right‐handed α‐helical state to positive Cotton effects. The pH profile of this Cotton effect is different from that of the peptide Cotton effects, which reflect the helix‐random coil equilibrium. The data are interpreted in terms of preferential breakdown of the α‐helix in the neighborhood of the tyrosine residues. An ultraviolet difference spectrum in the tyrosine absorption bands is generated at the low pH extreme of the conformational transition, the absorbance change being largely complete at a pH at which the other optical parameters have only begun to change. A possible explanation is the formation of a hydrogen bond between the phenolic hydroxyl and a carboxylate, the pK of which is lowered by the hydrogen bonding. An alternative explanation is the freezing of side‐chain rotations at a pH below the onset of the helix‐random coil transition, when the degree of side chain ionization approaches zero. Some support for the latter scheme comes from the splitting of side‐chain methylene proton resonances, indicating partial immobilization, as well as small changes in chemical shift of tyrosine ring protons in the pH (or pD) region in which the difference spectrum appears.

Lactoferrin and transferrin: A comparative study

The optical and electron paramagnetic resonance (EPR) spectroscopic parameters which characterize the two metal-binding sites of lactoferrin are strikingly similar to those of transferrin. Ultraviolet difference spectra indicate the involvement of tyrosyl residues in iron binding, while EPR studies of the Cu(II)-lactoferrin-bicarbonate complex demonstrate the presence of at least one nitrogen ligand at each metal-binding site. However, the K 1 for Fe(III) binding as obtained by equilibrium dialysis and corrected for H + effects, is 300 times larger for human lactoferrin (measured near pH 6.4) than for human transferrin (measured near pH 6.7). Fe(III) binding to both proteins appears to involve equivalent and non-interacting sites. The disulfide structure of lactoferrin and transferrin also appear to be similar, as shown by gel-filtration studies. This, together with the nearly identical molecular weights of the proteins, their single-chain nature, metal-binding sites, corroborate the suggestion that the transferrins and lactoferrins constitute a single class of proteins evolving from a common ancestral protein.

Food Chemical Study of Soybean Proteins

The conformational change of the 11S component of soybean proteins in acid was measured by ultraviolet difference spectra, optical rotatory dispersion and circular dichroism. Below pH 4, the change of tertiary structure that the tyrosine residues were exposed and the tryptophan residues were barried into the molecule was recognized, and, between pH 2 and 0.64, the change was attained to maximum. The secondary structure, β-structure, of the 11S component was almost stable at pH 1.22.

Food Chemical Study of Soybean Protein

The conformational change of the 11S component of soybean proteins in acid was measured by ultraviolet difference spectra, optical rotatory dispersion and circular dichroism. Below pH 4, the change of tertiary structure that the tyrosine residues were exposed and the tryptophan residues were barried into the molecule was recognized, and, between pH 2 and 0.64, the change was attained to maximum. The secondary structure, β-structure, of the 11S component was almost stable at pH 1.22.