Difference Spectral Measurements Research Articles

Neutron diffraction study of concentrated aqueous lithium benzoate solutions

Time-of-flight neutron diffraction and infrared (IR) double difference spectral measurements have been carried out on aqueous 4 mol% lithium benzoate solutions. The neutron diffraction with isotopic substitution technique has been applied to both lithium and benzyl-hydrogen atoms within the benzoate ion in order to obtain information on the hydration structure around both the lithium ion and the benzyl-hydrogen atom within the benzoate ion. The observed first-order difference functions, Δ Li( Q) and Δ H inter( Q), were analyzed by the least squares fitting procedure. The number of water molecules within the first hydration shell of the Li + was determined to be 5.3(1) with the Li +···O and Li +···D distances of 1.95(1) and 2.58(1) Å, respectively. A well-defined second hydration shell involving 6.0(1) water molecules, has been found and characterized by the Li +···O and Li +···D distances of 4.12(1) and 4.73(1) Å, respectively. The first hydration shell of the benzyl-hydrogen atoms (H b) within the benzoate ion was found to consist of 1.0(1) water molecule with the average H b···D 2O intermolecular distance of 2.78(1) Å. Results of the IR double difference spectra exhibited that the number of water molecules within the first hydration shell of Li + is 5.7(1), in reasonable agreement with that obtained from the neutron diffraction data. The hydration number of the carboxyl group within the benzoate ion was determined to be 4.9(1) from the IR difference spectra.

Unfolding-refolding behaviour of chicken egg white ovomucoid and its correlation with the three domain structure of the protein

The urea and heat-induced unfolding-refolding behaviours of chicken egg white ovomucoid and it four fragments representing domains I, II + III, I ÷ II and III were systematically investigated in 0.06 M sodium phosphate buffer (pH 7.0) by difference spectral measurements. The effect of temperature on ovomucoid and its fragments was also studied in 0.05 M sodium acetate buffer (pH 5.0) and in presence of 2 M urea at pH 7.0. Intrinsic viscosity data showed that ovomucoid and its different fragments did not lose any significant amount of their structure under mild acidic conditions (pH 4.6). Difference spectral results showed extensive disruption of the native structure by urea or temperature. Isothermal transitions showed single-step for domain I, domain I + II and domain III, and two-step having one stable intermediate, for ovomucoid and its fragment representing domain II + III. However, the presence of intermediate was not detected when the transitions were studied with temperature at pH 7.0. Strikingly, the single-step thermal transitions of ovomucoid and its fragment representing domain II + III, became two-step when measured either at pH 5.0 or in presence of 2 M urea at pH 7.0. Analysis of the equilibrium data on urea and heat denaturation showed that the second transition observed with ovomucoid or domain II + III represent the unfolding of domain III. The kinetic results of ovomucoid and its fragments indicate that the protein unfolds with three kinetic phases. A comparison of three rate constants for the unfolding of intact ovomucoid with that of its various fragments revealed that domain I, II and III of the protein correspond to the three kinetic phases having rate constants 0.456, 0.120 and 0.054 min −1, respectively. These data have led us to conclude: (i) the unusual stability of ovomucoid towards various denaturants, including temperature, is due to its domain III, (ii) initiation of the folding of the ovomucoid molecule starts from its NH 2-terminal region which probably provides the nucleation site for the formation of the subsequent structure and (iii) domains I and II have greater mutual recognition between them as compared to the recognition either of them have with domain III.

R 76713 and enantiomers: Selective, nonsteroidal inhibitors of the cytochrome P450-dependent oestrogen synthesis

The triazole derivative, R 76713 and its enantiomers R 83839(−) and R 83842(+) are effective inhibitors of the aromatization of androstenedione. For human placental microsomes, the (+) enantiomer (R 83824) is about 1.9- and 32-times more active than the racemate ( ic 50 2.6 nM) and the (−) enantiomer, respectively. R 83842 is about 30- and 1029-times more active than 4-hydroxy-androstene-3, 17-dione and aminoglutethimide. This potency might originate from its high affinity for the microsomal cytochrome P450 (P450). Indeed, R 83842, compared to R 76713 and R 83839, forms a more stable P450-drug complex. Difference spectral measurements indicate that the triazole nitrogen N-4 coordinates to the haem iron. The reversed type I spectral changes suggest that R 76713 is able to displace the substrate from its binding place and the stable complex formed in particular with the (+) enantiomer suggests that its N-1-substituent occupies a lipophilic region of the apoprotein moiety. Kinetic analysis implies that there is a competitive part in the inhibition of the human placental aromatase by R 76713. The K i , values for R 76713, R 83842 and R 83839 are 1.3 nM, 0.7 nM and 18 nM, respectively. These results are indicative of stereospecificity for binding. Up to 10 μM, R 76713 and its enantiomers have no statistically significant effect on the regio- and stereoselective oxidations of testosterone in male rat liver microsomes. All three compounds have no effect on the P450-dependent cholesterol synthesis, cholesterol side-chain cleavage and 7α-hydroxylation and 21-hydroxylase. At 10 μM, R 76713 has a slight effect on the bovine adrenal 11β-hydroxylase. This effect originates mainly from R 83839, the less potent aromatase inhibitor. On the other hand, the inhibition of the 17,20-lyase of rat testis observed at concentrations ⩾ 0.5 μM, originates rather from R 83842. However, 50% inhibition is only achieved at 1.8μM R 83842, i.e. at a concentration about 1300-times higher than that needed to reach 50% inhibition of the human placental aromatase.

Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants.

Characteristics and properties of the unfolding free energy change, delta G degrees N-U, as determined by the linear extrapolation method are assessed for the unfolding of phenylmethanesulfonyl chymotrypsin (PMS-Ct). Difference spectral measurements at 293 nm were used to define PMS-Ct unfolding brought about with guanidinium chloride, urea, and 1,3-dimethylurea. All three denaturants were shown to give identical extinction coefficient differences (delta epsilon N-U) between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of delta epsilon N-U on denaturant supports the linear extension of pre- and postdenaturational base lines into the transition zone, allowing evaluation of unfolding equilibrium constants based on the two-state assumption. An expression, based on the linear extrapolation method, was used to provide estimates of delta G degrees N-U for the three denaturants using nonlinear least-squares fitting of the primary data, delta epsilon versus [denaturant]. The three delta G degrees N-U values were identical, within error, suggesting that the free energy change is a property of the protein system and independent of denaturant. It is suggested that the error in delta G degrees N-U determined from use of the linear extrapolation method is significantly larger than commonly reported in the literature.

Synthesis and biological properties of actinomycin D chromophoric analogues substituted at carbon 7 with aziridine and cyclopropyl functions.

The growing importance of functionalized tricyclic rings, e.g., cyclopropyl and aziridine, in numerous organic biomolecules led us to develop syntheses of novel actinomycin D (AMD) analogues substituted with aziridine and cyclopropyl functions. Reaction of 7-hydroxyactinomycin D with 1-aziridineethyl iodide and bromomethylcycloporopane afforded the desired 7-[2-(1-aziridinyl)ethoxy] and cyclopropylmethoxy analogues, respectively. Calf thymus DNA binding of these analogues was comparable to that of AMD as examined by UV-vis difference spectral measurements, CD techniques, and relaxation of supercoiled closed circular SV40 DNA, indicating an intercalative mode of binding to the DNA duplex. Thermal denaturation of DNA experiments employing higher temperatures than room temperature exhibit a thermal lability of the DNA analogue complexes, suggestive of a probable covalent bond formation with DNA bases. The analogues were found to be 1/4-1/40 as cytotoxic to human lymphoblastic CCRF-CEM leukemia and B16 melanoma cells in vitro as AMD, with ID50 values in the nanomolar concentration range.

Domain II + III of bovine serum albumin: Isolation and its characterization

Some properties of a fragment of bovine serum albumin containing residues 184–582 of the protein sequence, produced by cyanogen bromide cleavage, have been reported. Urea-induced difference spectra of the fragment showed considerable exposure of aromatic chromophores by 8 M urea. Reversible unfolding of the fragment by urea, as followed by difference spectral measurements at 30°C, pH 7.0, occurred in two distinct steps involving at least 3 major conformational states, namely the native (N), intermediate (X) and completely denatured (D) states. The co-operativity values for the two transitions, N⇌X and X⇌Dwere found to be 4.0 and 16.4, respectively. Analysis of the data on bilirubin binding to bovine serum albumin and its fragment suggested that the fragment retains significant amount of its native structure. However, hydrodynamic parameters such as Stokes radius (3.f14 nm), diffusion coefficient (6.98 × 10−7cm2/s) and frictional ratio (1.32) obtained by analytical gel chromatography as well as intrinsic viscosity (4.31 ml/g) indicates some asymmetry in the fragment molecule.

Interaction of urea with the high molecular weight protein fraction, carmin, from safflower seed (Carthamus tinctorius)

The effect of urea on the high molecular weight protein fraction, carmin, from safflower seed Carthamus tinctorius L, was investigated by the techniques of sedimentation velocity, viscosity, fluorescence and difference spectral measurements, partial specific volume and circular dichroism. The protein underwent dissociation and denaturation simultaneously in the presence of increasing concentration of urea. The protein dissociated to a 2S component and in the process, the buried and exposed residues of tyrosines and tryptophans were perturbed. At 8 M urea concentration, the protein existed completely as a random coil; nearly 535 mol of urea were bound per mol of protein. The results indicated that the subunits of carmin appears to be held principally by noncovalent interactions and are dissociated/denaturated by increasing concentration of urea, leading to the destabilization of the native oligomer.

Inhibition of estrogen synthetase (aromatase) by 4-cyclohexylaniline.

4- Cyclohexylaniline , a structurally simple analog of the drug aminoglutethimide [d,l-3-(4-aminophenyl)3-ethyl-2, 6- piperidinedione ], was found to be an effective inhibitor of the aromatization of testosterone and androstenedione. With human placental microsomes, 4- cyclohexylaniline was a more potent aromatase inhibitor than d-aminoglutethimide. For androstenedione and testosterone aromatization, competitive inhibition by 4- cyclohexylaniline was observed; a Ki value of 0.14 microM was found with both substrates. A Ki value for d-aminoglutethimide inhibition of androstenedione aromatization of 0.3 microM was obtained. Kinetic analysis of the simultaneous inhibition by 4- cyclohexylaniline and d-aminoglutethimide suggests that both compounds bind to the same site on the enzyme. 4- Cyclohexylaniline and d-aminoglutethimide were also tested for inhibition of androstenedione aromatization in human and rat ovarian microsomes. With the human aromatase, both inhibitors exhibited approximately the same effectiveness; with rat aromatase, however, d-aminoglutethimide was more potent. 4- Cyclohexylaniline and d-aminoglutethimide were also assayed for their inhibition of cytochrome P-450-catalyzed cholesterol side-chain cleavage. When the enzyme from human placenta was used, 4- cyclohexylaniline was 24-fold less effective than d-aminoglutethimide, and when the purified bovine adrenal enzyme was used, it was 16-fold less effective. Thus, 4- cyclohexylaniline exhibits inhibitory specificity toward aromatase. Difference spectral measurements using crude placental microsomes and cholate extracts of these microsomes show that binding of 4- cyclohexylaniline produces a type II spectral change; this is indicative of coordination of the arylamine to the heme-iron of the aromatase cytochrome P-450. Consistent with this spectral finding was the fact that blockade of the amino group of both 4- cyclohexylaniline and d-aminoglutethimide by acetylation resulted in essentially complete loss of inhibitory activity toward aromatase. These findings establish that the arylamine moieties of both 4- cyclohexylaniline and d-aminoglutethimide are essential for their inhibitory actions. The potent aromatase inhibition by 4- cyclohexylaniline indicates that neither the complex glutarimide ring nor the chiral 3-ethyl substituent of d-aminoglutethimide is required for high affinity binding of this class of inhibitor to aromatase P-450.

Free energy changes in denaturation of ribonuclease A by mixed denaturants. Effects of combinations of guanidine hydrochloride and one of the denaturants lithium bromide, lithium chloride, and sodium bromide.

The denaturation of ribonuclease A by guanidine hydrochloride, lithium bromide, and lithium chloride and by mixed denaturants consisting of guanidine hydrochloride and one of the denaturants lithium chloride, lithium bromide, and sodium bromide was followed by difference spectral measurements at pH 4.8 and 25 degrees C. Both components of mixed denaturant systems enhance each other's effect in unfolding the protein. The effect of lithium bromide on the midpoint of guanidine hydrochloride denaturation transition is approximately the sum of the effects of the constituent ions. For all the mixed denaturants tested, the dependence of the free energy change on denaturation is linear. The conformational free energy associated with the guanidine hydrochloride denaturation transition in water is 7.5 +/- 0.1 kcal mol-1, and it is unchanged in the presence of low concentrations of lithium bromide, lithium chloride, and sodium bromide which by themselves are not concentrated enough to unfold the protein. The conformational free energy associated with the lithium bromide denaturation transition in water is 11.7 +/- 0.3 kcal mol-1, and it is not affected by the presence of low concentrations of guanidine hydrochloride which by themselves do not disrupt the structure of native ribonuclease A.

Free energy changes in ribonuclease A denaturation. Effect of urea, guanidine hydrochloride, and lithium salts.

The unfolding of ribonuclease A by urea, guanidine hydrochloride, lithium perchlorate, lithium chloride, and lithium bromide has been followed by circular dichroic and difference spectral measurements. All three abnormal tyrosyl residues are normalized in urea and guanidine hydrochloride (delta epsilon 287 = -2700), only two are normalized in lithium bromide and lithium perchlorate (delta epsilon 287 = -1700), and only one is exposed in lithium chloride solutions (delta epsilon 287 = -700). The Gibbs energies are 4.7 +/- 0.1 kcal mol-1 for urea- and guanidine hydrochloride-denaturation, 3.8 +/- 0.2 kcal mol-1 for lithium perchlorate-denaturation, and 12.7 +/- 0.2 kcal mol-1 for lithium chloride- and lithium bromide-denaturation of ribonuclease A. The latter results suggest that the mechanism of the unfolding process in urea and guanidine hydrochloride is quite different from that in lithium salts.

Regulation of Escherichia coli carbamyl phosphate synthetase. Evidence for overlap of the allosteric nucleotide binding sites.

Regulation of Escherichia coli carbamyl phosphate synthetase by UMP and IMP was examined in studies with various analogs of these nucleotides. Whereas UMP inhibits enzyme activity, the arabinose analog of UMP was found to be an activator. dUMP neither activates nor inhibits, but binds to the enzyme in a manner similar to UMP as evaluated by direct binding studies, sedimentation behavior, and ultraviolet difference spectral measurements. dUMP decreases inhibition by UMP and activation by IMP, but has no effect on activation by L-ornithine. The findings are in accord with the view that IMP and UMP bind to the same region of the enzyme; a possible general model for such overlapping binding sites is considered. Additional evidence is presented that inorganic phosphate can modulate regulation of the activity by nucleotides. Phosphate (and arsenate) markedly increase inhibition by UMP, decrease activation by IMP, but do not affect activation by L-ornithine. The extent of activation by IMP and by L-ornithine and that of inhibition by UMP are decreased when Mg2+ concentrations are increased relative to a fixed concentration of ATP. The findings suggest that the allosteric effectors may affect affinity of the enzyme for divalent metal ions as well as, as previously shown, the affinity of the enzyme for Mg-ATP.

Estimation of the free energy of stabilization of ribonuclease A, lysozyme, alpha-lactalbumin, and myoglobin.

The denaturation of ribonuclease A, lysozyme alpha-lactalbumin, and myoglobin by urea, guanidine hydrochloride, and guanidine thiocyanate has been followed with the use of difference spectral measurements. The free energy of stabilization (delta GH2OD) has been determined by the linear extrapolation of the free energy of denaturation to zero denaturant concentration. The values of delta GH2OD are 7.3 +/- 0.2, 8.9 +/- 0.1, 4.3 +/- 0.1;, and 7.9 +/- 0.2 kcal/mol at 25 degrees C for ribonuclease A (pH 7.0), lysozyme (pH 7.0), alpha-lactalbumin (pH 7.0), and myoglobin (pH 6.6), respectively. The dependence of the free energy of denaturation on concentration ranges from 0.88 to 2.08 kcal/mol.M for urea and from 1.27 to 4.22 kcal/mol.M for guanidine hydrochloride for four proteins. The ratio of this dependence in guanidine hydrochloride to that in urea may depend on the polarity of the amino acid residues in the unfolding unit.

Kinetic analysis of calcium binding to concanavalin A

The kinetics of calcium binding to concanavalin A was studied utilizing ultraviolet difference spectral measurements. The results show that calcium binds to the lectin in a biphasic process: a rapid and reversible phase, followed by a relaxation phase with a k obs of 0.012 sec −1. Kinetic measurements were used to calculate the association constant, K a , for calcium binding to concanavalin A of 2.7 x 10 4 M −1, in reasonable agreement with values obtained by equilibrium methods.

Occurrence and characterization of stable intermediate state(s) in the unfolding of ovomucoid by guanidine hydrochloride.

Reversible unfolding of ovomucoid by guanidine hydrochloride, as followed by viscosity and difference-spectral measurements at 25 degrees C, pH6, occurred in two distinct steps involving at least three major conformational states, namely the native, intermediate and completely denatured states, occurring respectively in 60mm-sodium phosphate buffer, 3.5m-guanidine hydrochloride and 6m-guanidine hydrochloride. The overall native conformation of ovomucoid, as indicated by its intrinsic viscosity (5.24ml/g) and gel-filtration behaviour, differs significantly from that of a typical globular protein. Exposures of tyrosine residues in native ovomucoid measured by difference spectroscopy following perturbation with glycerol, ethylene glycol and dimethyl sulphoxide were, respectively, 0.42, 0.56 and 0.57. Of the exposed phenolic groups only one titrated normally (pK(int.), 9.91, electrostatic-interaction factor, w, 0.04). Results on difference spectra, solvent perturbation, phenolic titration and intrinsic viscosity (7.4ml/g) taken together showed that, although ovomucoid in 3.5m-guanidine hydrochloride was significantly unfolded, it retained a degree of native structure, removable with 6m-guanidine hydrochloride. In the latter, all the six tyrosine residues were available for titration, and the intrinsic viscosity of ovomucoid increased to 9.4ml/g. Furthermore, the characteristic fine structures in circular-dichrosim spectra of ovomucoid, associated with the elements of native structure, were abolished in 6m-guanidine hydrochloride, suggesting that the completely denatured state is structureless and presumably behaves as a cross-linked random coil. The latter state has been shown by analysis of the results on guanidine hydrochloride-dependence of the transition, intermediateright harpoon over left harpoondenatured, to be less stable than the intermediate state under native conditions by about 46kJ/mol at 25 degrees C. Attempts have been made to interpret the above results in the light of available information on the amino acid sequence of ovomucoid.

The influence of lysolecithin on the complex formation between beta-lactoglobulin and kappa-casein.

SummaryUltraviolet difference spectral measurements and some circular dichroism data are reported on the systems (β-lactoglobulin (BLG)/soya-lysolecithin (sll)), (κ-casein (KC)/sll) and (BLG/KC/sll). Sll destabilizes the native conformation of BLG and KC and promotes the interaction between BLG and KC. The influence of sll: protein ratio and time of heating on the complex formation has been investigated.

Difference spectral method for determination of native G-actin concentration.

Abstract A relatively fast quantitative determination of the native G-actin content of purified G-actin preparations is described, based on difference spectral measurements of EDTA-denatured actin vs native G-actin. The changes at 291, 281, and 255 nm are linear with native G-actin concentrations and give absorbancy changes of −0.05, −0.06, and 0.10, respectively, for a 1 mg G-actin/ml solution in a 1 cm light path.

Calorimetric Determination of Denaturation Enthalpy for Lysozyme in Guanidine Hydrochloride

Abstract A procedure is described for direct calorimetric determination of denaturation enthalpies of proteins in the presence of denaturing agents. The method has been applied to the reversible transition of lysozyme between native and unfolded conformations in guanidine hydrochloride at 25°. The heat of pH-induced unfolding has been measured at various concentrations of guanidine HCl and found to be 30 kcal per mole under conditions of complete transition at 3 m guanidine HCl. The corresponding van't Hoff enthalpy was determined from difference spectral measurements at 301 nm as a function of temperature and found to be 29 kcal per mole. Agreement between the calorimetric and van't Hoff enthalpies indicates the absence of appreciably populated intermediate states between native and denatured forms of the protein.

The Formation of Diiodotyrosine in Iodinated Human Serum Albumin

Abstract Human serum albumin (HSA) has been allowed to react with varying amounts of iodine under mild conditions. Almost all of the tyrosyl residues of HSA may be converted to iodotyrosyl residues (primarily diiodotyrosyl) without producing significant changes in the conformation of the protein. Difference spectral measurements in urea show the disappearance of buried tyrosyl and the appearance of buried diiodotyrosyl residues; a difference spectral peak attributed to diiodotyrosyl residues in iodinated HSA is seen at 302 mµ. It is proposed that the partly nonaqueous environment of buried tyrosyl residues directs their preferential conversion to diiodotyrosyl residues.

Assessment of Conformational Changes in Low-Sulphur S-Carboxymethylkerateine from Wool

The effects of urea and guanidine hydrochloride on the ultraviolet absorption spectrum of the low-sulphur S-carboxymethylkerateine fraction of wool have been measured. In concentrated solutions of urea characteristic difference spectra were obtained with maxima of negative absorbance at 288, 280, and 240 miL. The addition of guanidine hydrochloride or an increase in temperature gave similar negative difference maxima at the higher wavelengths. Calculation of the extent of unfolding of the protein chains from the difference in absorbance at all three maxima showed that the unfolding was 50% complete at a urea concentration of about 1� 8M whereas a urea concep.tration of about 4� 3M was required to decrease the helix content by 50%. Similar measurements on components 7 and 8, the two major constituents of SCMKA, showed that a 50% decrease in helix content was obtained with 2�8M and O� 8M urea respectively whereas the corresponding values for 50 % unfolding assessed from difference spectral measurements were 2� 2M and 1� 2M urea respectively. It is suggested that the helical regions of components 7 and 8 aggregate specifically and that spectral measurements relate largely to non-helical portions of the chains.