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The phenylalanine-specific tRNAPhe from 13-day-old chick embryo muscle was purified. Its fluorescent and hydrophobic Y+ base was excised by mild acid treatment and characterized chromatographically and by its ultraviolet absorption and fluorescent spectra. The compound obtained after removal of the Y+ base from chick embryo tRNAPhe was characterized. 1. Chick embryo t RNAPhe looses by the removal of the Y+ base its phenylalanine acceptor capacity. 2. In this state it has a reduced sedimentation coefficient of 2.8 S. a low Tm value (22 °C), only rudimentary hyperchromicity, but unchanged electrophoretic properties. 3. The compound obtained after removal of the Y+ base from chick embryo tRNAPhe regains normal melting behaviour and phenylalanine acceptor capacity when exposed to special renaturation conditions. 4. The Km of the renatured form is 0.25 × 10-6 M and V = 220 pmol × min-1 × ml-1 as compared tO Km = 0.11 × 10-6 M and V = 370 pmol x min-1 × ml-1 for the native chick embryo tRNAPhe. [ABSTRACT FROM AUTHOR]

Complexes of cytochrome c and of an undecapeptide of the protein have been studied by means of Mössbauer spectroscopy in order to obtain information concerning the active heme site. The variation &the quadrupole splitting with the temperature pewits an evaluation of the distortions from octahedral symmetry around the iron ion in the heme group, and also an estimation of the spin-orbit constant. The effect of the different ligands such as CN-, F-, imidazol, etc., is discussed. [ABSTRACT FROM AUTHOR]

The effect of calcium ions on steroid-induced difference spectra of cytochrome P-450, and on pregnenolone formation, has been studied in rat adrenal mitochondria. In the presence of phosphate, addition of calcium chloride to suspensions of these mitochondria resulted in an increase in the inverted type-I difference spectrum induced by pregnenolone or methylandrostenediol and a disappearance of the type I spectrum induced by 25-hydroxycholesterol. The difference in the inverted type I spectrum between mitochondria from ether-stressed animals and those given cycloheximide was abolished. Cholesterol side-chain cleavage was increased, providing an NADPH generator was used as source of reducing equivalents. The difference in side-chain cleavage activity between mitochondria from ether-stressed animals and those given cycloheximide, was maintained. In the absence of phosphate, addition of calcium chloride again resulted in a loss of the type I difference spectrum induced by 25-hydroxycholesterol, but this time there was almost no change in the inverted type I spectrum induced by pregnenolone in mitochondria from rats subjected to ether anaesthesia, and the change in cycloheximide-treated rats was only about 50% of the change in the 25-hydroxycholesterol-induced difference spectrum. These observations are interpreted in terms of interconversions of the various forms of cytochrome P-450 associated with side-chain cleavage proposed to exist in intact adrenal mitochondria. Factors such as bovine serum albumin and EDTA, which increase respiratory control in these mitochondria, also stimulate cholesterol side-chain cleavage activity, as do magnesium ions, but without altering the magnitude of the steroid-binding spectra.

The following fluorescence parameters have been investigated: emission and excitation spectra, relative quantum yields, polarization values and lifetimes of the apo- and holoenzyme of lipoamide dehydrogenase. Furthermore, fluorescence perturbation techniques have been used to characterize qualitatively the tryptophan and flavin environment in the protein. Also emission spectra at low temperature have been recorded to examine changes occurring on strong immobilization of the enzyme. From lifetimes and quantum yields of the protein fluorescence it can be concluded that the molecular structures of the two apoenzymes, which can be prepared from this enzyme, are different. The quenched protein fluorescence of the holoenzyme is characterized by a very short fluorescence lifetime due to radiationless energy transfer from tryptophan to flavin. Excitation polarization spectra of flavin and tryptophan fluorescences show that the chromophores are rigidly bound to the protein. Luminescence studies at liquid nitrogen temperatures reveal that the triplet state of the tryptophanyl-residues is not involved in energy transfer to the flavin. Collisional fluorescence quenching with KI shows in the dimeric holoenzyme, in contrast with the free molecules, only a small reduction in fluorescence intensity of tryptophanyl and flavin residues. Charge effects might be responsible for these observations. It can be derived from the experimental results together with the indication that only one of the two tryptophans is transferring its excitation energy to the flavin, that the distance between donor and acceptor is 1.3-1.6 nm.

A study of reaction mechanisms of indole-3-acetate degradation by various peroxidases, in the absence of added hydrogen peroxide, has been performed employing stopped-fow and lowtemperature spectroscopic techniques. At pH 6 and for high [substrate]/[enzyme] ratios, peroxidase compound II is formed and is stable for several minutes. However, this enzyme compound becomes visible only after the transient appearance of ferroperoxidase and of compound III. This reaction process is apparently linked with the formation of 3-methyleneoxindole as the major end-product of indole-3-acetate degradation. On the other hand, when indole-3-acetate and peroxidase in stoichiometric concentrations are mixed at pH 4, only ferroperoxidase and compound III are formed and the process appears to be linked with the production of indole-3-aldehyde. Stopped-flow experiments show that indole-3-acetate is rapidly bound on peroxidase, this rapid binding step being followed by a slow one-electron transfer between indole-3-acetate and peroxidase. This reduction of ferriperoxidase is blocked at low temperature (-20 °C), whereas in these experimental conditions, peroxidation of indole-3-acetate in the presence of added hydrogen peroxide still occurs. Moreover, when compound III reacts with indole-3-acetate at low temperature (-15 °C}, this compound is regenerated as long as oxygen is present in the reaction medium. Neither ferroperoxidase nor compound I appear during this process. Most, if not all of the compound III is formed by oxygenation of the ferrous peroxidase. This compound III can react with an equimolar amount of indole-3-acetate to give ferrous peroxidase and indole-3-aldehyde. Moreover, compound III can react with two indole-3-acetate molecules leading to compound II formatiom. Because only small amounts of the organic peroxide derived from indole-3-acetate are present in the reaction medium, most of the compound II is formed by a two-equivalent reduction of... [ABSTRACT FROM AUTHOR]

Lactate dehydrogenase A4 from possum, chicken, and sardine muscle extracts and lactate dehydrogenase B4 from chicken heart, and sardine muscle extracts have been purified. Specific activity determinations confirmed previous studies showing the A4 isoenzyme to be approximately twice as efficient as the B4 isozyme and fish lactate dehydrogenase A4 to be considerably more active than the corresponding enzyme in warm-blooded animals. Spectrophotometric analyses demonstrated striking similarities in ε2801 mg/ml e a values, which reflected a uniform tryptophan content (5 residues lactate dehydrogenate subunit) in the enzyme, is analyzed. Sheep antisera were prepared against each isoenzyme and used in immunochemical titration experiments to measure the extent of inhibition, precipitation and interaction of the lactate dehydrogenase isozymes by the antisera. Lactate dehydrogenase antibodies differed in their immunochemical specificities according to the nature and source of the antigen. For example, the relative affinities of antiserum to possum A4 isozyme for the enzymes was in the order, possum A4>, chicken A4>, sardine A4..., sardine B4, chicken B4 which compared with the affinities for antichicken B4, chicken B4>, sardine B4... sardine, chicken and possum A4. In contrast to the results of other workers, isozymes A4 and B4 were shown to be immunochemically related. This observation is attributed to the higher degree of sensitivity in recognizing enzyme-antibody interactions using gel zymogram/immunochemical technique. The antisera were also used in investigating the immunochemical properties of isozymes A4, A4', B4, B4' and E4 from trout, and isozymes A4, B4 and C4 from pigeon, kangaroo and rat-tissue extracts. These vertebrate lactate dehydrogenase isozymes shared common antigenic determinants which reflected differing degrees of sequence homology and a common evolutionary origin. [ABSTRACT FROM AUTHOR]

Interactions of sperm whale ferrimyoglobin with the cationic detergent laurylpyridinium chloride in aqueous medium were studied by measurement of spectral changes in the Soret band of the prosthetic group. Spectrophotometric titrations were carried out in 0.02 M sodium phosphate buffer pH 7.1 at 25°C in the concentration ranges of 1 nnM-1 μM myoglobin and up to 10 mM laurylpyridinium chloride and also at various NaCl concentrations, up to 0.5 M. A thermodynamic analysis was carried out assuming in all-or-none interaction between the hemoprotein and the detergent. The apparent stoichiometric coefficients for the association of the detergent with the protein and the average standard free energy changes of the interactions were evaluated. The thermodynamic data obtained correspond to values characteristic for hydrophobic interactions. The dependence of the spectral transition curves on total myoglobin concentration found in the present system enabled an evaluation of equilibria involving dissociation of the prosthetic group from the ferrimyoglobin by the ac~ien of the cationic detergent. Direct experimental evidence obtained for this process by chromatography has been presented previously. This leads to interpretation of the spectral effects observed as a result of association between ferriheme and laurylpyridinium chloride molecules. [ABSTRACT FROM AUTHOR]

Specific interaction between α-NADH and glutamate dehydrogenase is demonstrated by difference spectroscopy, circular dichroism and fluorescence measurements. Quantitative binding studies in the preparative ultracentrifuge yield six identical α-NADH binding sites per oligomer with a dissociation constant of 20 μM. Evidence for six to eight additional, very weak α-NADH binding sites is presented. Excess ADP prevents the binding of α-NADH to the tight binding sites, indicating competition of the two nucleotides. In the enzymatic reaction, α-NADH is inactive as a coenzyme; however, like ADP, it is an activator of the reductive amination reaction of 2-oxoglutarate. α-NADH reduces the self-inhibition of high β-NADH concentrations and thus again parallels the effects caused by ADP. Contrary to β-NADH or β-NADH plus GTP, α-NADH or α-NADH plus GTP does not affect the dissociation-association equilibrium of glutamate dehydrogenase. The results are consistent with the assumption of six binding sites for α-NADH, which are identical with the ADP binding sites and most probably the six non-active β-NADH binding sites. [ABSTRACT FROM AUTHOR]

The interaction of αs1-casein with calcium ions at pH 7.0 has been studied, and it is shown that there is a stepwise increase in the fluorescence of the tryptophan residues of the protein as increasing amounts of Ca2+ are added, leading to eventual precipitation of the calcium-caseinate complex. The first of these steps appears to result from a conformational change induced by Ca2+, without which the protein cannot precipitate. Further steps arise from interaction between protein molecules to produce particles of about 120 nm in diameter, then final aggregation of these particles to give a precipitate. All of these changes result in changes in the fluorescence emission spectra of one or both tryptophan residues in the casein, although most of the change in fluorescence behaviour in the first step of the interaction probably arises from tryptophan-164 in the protein: [ABSTRACT FROM AUTHOR]

The chemical shift of the phosphorus resonance of various nucleotides has been studied as a function of pH in the absence and presence of ribonuclease A. The 31P-signal of the nucleotides shifts upfield upon protonation of the phosphate moiety. Thus titration curves and pK values are obtained when the 31P chemical shift is plotted against the pH value. The titration curves are analyzed in terms of interactions of the phosphate group with the base or ribose moieties of the nucleotide itself or with amino-acid side chains of the active site of ribonuclease A. The chemical shift of the phosphorus magnetic resonance of cytidine 2′-phosphate when this nucleotide is bound to ribonuclease A resembles that of the dianionic form of the free compound. Proton magnetic resonance studies indicated that the histidine residue 119 of ribonuclease A is bound to the phosphate group in enzyme-nucleotide complexes [4]. This is now confirmed, since the apparent pK value of the phosphate group of these complexes is the same as the pK value of histidine.ll9 determined by proton magnetic resonance spectroscopy. Possible arrangements of the hydrogen bonds between the imidazole group of histidine-ll9 and the phosphate group are discussed. From linewidth broadening due to chemical exchange, an average life time of the complex of cytidine 2′-phosphate with ribonuclease A is derived. [ABSTRACT FROM AUTHOR]

A number of peptide substrates of the general structure Ac-Lxn … -Lx2-Lx1-Gly-N'H2, have been synthesized and their α-chymotrypsin-catalyzed hydrolyses studied. The acylation rate constants, k23 (= kcat), and the dissociation constants of the enzyme-substrate complexes, KEA (= Km), have been determined using a modified pH-stat and a numerical method for the acquisition and processing of the data. On the basis of these constants a quantitative relationship between the peptide structure N-terminal to the cleaved bond and reactivity has been determined. The results are shown to be consistent with the enzyme-substrate interaction scheme proposed in 1971 by Segal et al. (Biochemistry 10, 3728). A comparison of the k23/KEA values indicates that the influence of a single structural change on the overall reactivity is virtually independent of the nature of the remainder of the substrate. In addition a comparison of the k23 and KEA values shows that, in general, changes in substrate structure are mainly reflected by changes in k23 rather than in KEA. A few exceptions have been found: KEA or KEA and k28 change when glycine is introduced at the x2 position, when this glycine is replaced by alanine or when alanine is introduced at the x3 position. [ABSTRACT FROM AUTHOR]

The environment of the active site of creatine kinase has been investigated by means of three chromophoric reagents :2-hydroxy-5-nitrobenzyl bromide, 2-methoxy-5-nitrobenzyl bromide and 2-(dansylamino)ethyl monophosphate. 1. The first two reagents are incorporated at the essential thiol groups. The effect of pH on the spectra of the 2-hydroxy-5-nitrobenzyl-labelled creatine kinase suggests that this reagent lies inside the active site near an ionized residue, while the 2-methoxy-5-nitrobenzyl group is apparently exposed to the medium. Conformational changes induced by the binding of different ligands, including nucleotide substrates or anions, produce only perturbations of the 2-hydroxy-5-nitrobenzyl chromophore. 2. The 2-(dansylamino)ethyl monophosphate (which is really an analogue of the nucleotide substrate) interacts with the nucleotide site of creatine and arginine kinases in a relatively hydrophobic environment. [ABSTRACT FROM AUTHOR]

It has been found that fluorescein dimercuric acetate acts as a potential inhibitor of transaminase. Although the holoenzyme was inhibited also by fluorescein isothiocyanate the latter fluorochrome was found to be a rather specific inhibitor of the apoenzyme. The interactions involved are fast, the induced effect reaching its maximum value in less than 1 min. The isolated fluorescein dimercuric acetate or fluorescein isothiocyanate and transaminase or apotransaminase complexes suggest that the fluorochromes reacts specifically with two active sulfhydryl or amino groups of the enzyme, respectively. The fluorescence intensity of the fluorochromes is substantially quenched during their interaction with transaminase and apotransaminase. A red shift in the absorption spectrum maximum from 498 nm to 512 nm was also observed when fluorescein dimercurie acetate solutions were titrated with transaminase or apotransaminase. The recorded shift for fluorescein isothiocyanate in the fluorescein-isothiocyanate · apotransaminase complex was from 493 nm to 502 nm, respectively. In contrast to the previous spectral changes a blue shift in the spectrum maximum from 498 nm to 478 nm was recorded during the stoichiometric interaction of fluorescein dimercurie acetate with cysteine. Comparative studies concerning the reactivity of fluorescein dimercuric acetate or fluorescein isothiocyanate with transaminase, apotransaminase and cysteine or lysine suggest that the alterations observed in the spectral and fluorescence properties of the fluorochromes during their interaction with transaminase and apotransaminase arose from their complexation with specific sulfhydryl groups of the enzyme. These groups should be located in a critical hydrophobic area of the transaminase molecule since their interaction abolishes the catalytic activity of the enzyme and alters the spectral and fluorescence properties of the fluorochromes in the same way as low polarity solvents. These sulfhydryl and amino groups might also play a functional role since they interact faster with fluorescein dimercuric acetate and fluorescein isothlocyanate than the sulfhydryl group of cysteine or the amino group of lysine. Quantitative parameters of the data obtained by conventional techniques and stopped-flow spectroscopy during the interaction of the enzyme with the fluorochromes are also given. [ABSTRACT FROM AUTHOR]

The difference spectrum between oxidized and reduced fungal laccase at pH 5.5 shows an absorption band at 330 nm in addition to the visible absorption band at 610 nm. The 610 nm band and the 330 nm band are reduced together in a linear fashion with the addition of 3.5 electron equivalents of ascorbate. Titration of laccase in the presence of 3 mM fluoride leads to a differentiation of the electron- accepting sites in the molecule. The 610 nm band is found to be associated with a single electron- accepting site and the 330 nm band is associated with a two electron-accepting site. Electron paramagnetic resonance analysis during the titration indicates that neither the Type 1 Cu2+ (the ‘blue’ Cu2+) nor the Type 2 Cu2+ (the ‘non-blue’ Cu2+) are associated with the 330 nm band. Titration of laccase at pH 8.3 with quinol also associates the 330 nm band with a two electron-accepting site. Preliminary stopped-flow kinetic measurements on the absorbance changes at 330 nm and 610 nm are described and the results suggest an involvement of the component responsible for the 330 nm band in the catalytic reaction. The nature of the component responsible for the 330 nm band is discussed in the light of these results and our previous titration results. The findings are consistent with the 330 nm band being associated with the two additional copper atoms in the molecule and agree with our previous suggestion that these copper atoms exist as an electron-accepting Cu2+—Cu2+ pair in the oxidized protein. [ABSTRACT FROM AUTHOR]

Changes induced on addition of the cofactor, NAD, to glyceraldehyde-3-phosphate dehydrogenase from yeast have been investigated by hydrodynamic and spectroscopic techniques. As shown by light-scattering and sedimentation and diffusion studies, the tetrameric structure of the holoenzyme persists under all but strong dissociating conditions: there is thus no change in the quaternary structure on binding. On the other hand, landing is accompanied by an increase of some 40/0 in the sedimentation coefficient, which is too large to he explicable in terms of the increase in particle weight by bound ligand. A conformational change is also observed by optical rotatory dispersion and circular dichroism measurements. The magnitude of the Cotton effect associated with the aromatic chromophores of the protein, near 280 nm, increases, whereas the rotation in the neighbourhood of 233 urn undergoes a small diminution as the cofactor is added. it is shown that these changes are conformational in origin, rather than a consequence of WAD attachment per 89. Increasing the temperature in the range 2O-4O° leads to a transition from non-cooperative to cooperative binding of NAD to the apoenzyrne. At 40°, where NAT) binding is weakly sigmoidal, the change in optical rotation is likewise sigmoidal and parallels the increase in the sedimentation coefficient. At 20° both the uptake of WAD and the structural changes arc hyperbolic, in the cooperative situation the data are shown to be consistent with the two-state "concerted" model for ligand-binding and incompatible with a sequential mechanism. [ABSTRACT FROM AUTHOR]

Protein B2, one of the two non-identical subunits of ribonucleoside diphosphate reductase from Escherichia coli B, contains two atoms of inorganic iron per molecule of protein. The metal could be removed from the protein either by prolonged dialysis against 8-hydroxyquinoline or by precipitation with acid ammonium sulphate. This resulted in loss of enzyme activity which was recovered after treatment of the apoprotein with Fe2+ or Fe3+ but not with any other metals tested. Removal of iron did not change the behavior of the apoprotein on sucrose gradient centrifugation or on polyacrylamide gel electrophoresis as compared to native protein B2. No "labile sulphide" could be demonstrated in protein B2. Protein B2 had a characteristics spectrum with a very sharp peak at 410 nm, a broader one around 360 nm and steep shoulder at approximately 325 nm. The two peaks disappeared on removal of iron and reappeared on reactivation of the enzyme with the metal. When protein B2 was treated with hydroxyurea or hydroxylamine the peak at 410 nm (but not that at 360 nm) disappeared in parallel with enzyme activity, Such treatment did not result in the removal of iron. It is suggested that the very sharp peal at 410 nm originates from an unidentified component of protein B2. Furthermore, this component, together with iron, may be functional equivalent in the E. coli system of cobaminde coenzyme in ribonucleoside triphosphate reductase of Lactobacillus leichmannii. [ABSTRACT FROM AUTHOR]

A series of linear and cyclic derivatives of histidyl-tryptophan was synthesized for the study of intramolecular indole-imidazolium interaction. The absorption spectrum and the fluorescence intensity of each of the peptides were examined in aqueous solution when the imidazole side chain was protonated (pH < 4) and uncharged (pH > 9). The absorption spectra of the protohated peptides show hypochromism around 280 mμ, whereas at shorter wavelengths hyperchromism appears. The hyperchromism is most, intense around 240 mμ and it is attributed to the charge-transfer band of the intramolecular indole-imidazolium complex. The fluorescence intensity of all tested histidyl-tryptophan peptides drops markedly with protonation of the imidazole side chain. The change in fluorescence intensity with pH superimposes to the potentiometric titration curve of the imidazole residue. From the preceeding results it may be concluded that (a) the electron affinity of imidazolium is smaller than that of the positively charged nicotinamide by 1.2 eV and (b) the indole-imidazolium complex is not of a sandwich type. It is suggested that the charge-transfer bond of this complex is located between the C-2-C-3 bond of the indole and the plus charge center of the imidazolium. [ABSTRACT FROM AUTHOR]

It is shown that the perturbation of the ultraviolet absorption spectra of the tyrosine and tryptophan chromophores in aqueous solutions by addition of solvents of lower polarity leads not only to red shifts but also to changes in peak intensity, and oscillator strength. The effect is much greater in tyrosine than in tryptophan, and can be as large as 30% (transfer from water to ethanol). It is shown that observed difference spectra can be reasonably reproduced by the addition of two calculated curves, one corresponding to a shift, the other to a change in intensity, i. e. an admixture of shift difference spectrum and absolute spectrum. Similar intensity changes are seen to accompany the denaturation of several proteins. The changes of the characteristic parameters of difference spectra with the concentration of perturbant are analyzed. The relation between the difference spectra generated by aromatic chromophores near 230 mμ. and those m the 280 mμ. region are considered, and their relative magnitudes have been estimated for a variety of perturbants. It is shown that in denaturation. of a number of proteins, the difference spectra near 230 mμ cannot be satisfactorily accounted for solely in terms of the model compounds containing aromatic chromophores. Possible contributions to difference spectra of proteins in this region are considered. [ABSTRACT FROM AUTHOR]

The dependence on pH of the disproportionation of the flavosemiquinone has been investigated by ESR For 3-alkylated lumiflavin [R(3)=CH2COO- in aqueous solution p&Kdot; (for the dissociation of the neutral flavosemiquinone) is 8.4; pKred is 6.3. Hydrolytic ring cleavage in position 2,8,8'-dimerization, and 10-dealkylation interfere with the formation of the flavosemiquinone anions However, in non-aqueous dimethylformamide and with stoichiometric amounts of strong base (t-BuOK), 100% flavosemiquinone anion is formed without interfering side reactions. Its light absorption spectrum is practically identical to that of the "red flavoprotein radical". From the ESR spectra the individual isotropic hyperfine coupling constants have been determined. [ABSTRACT FROM AUTHOR]

Weak bases, such as the acridine and the aminophenazine dyes, which undergo protonation in the chromophoric group, are actively bound in sonicated fragments. On the other hand strong bases such as the divalent cations, the safranine and the cyanine dyes, which carry a permanent charge, are actively bound in intact mitochondria. In the latter case the process results in stimulation of the respiration, H+ ejection, and a large change in absorbance, which is attributed to a stacking of the dyes. The effects are inhibited or reversed by uncouplers, and insensitive to nigericin? Four alternatives are considered: accumulation in the inner aqueous space; binding to matrix proteins; binding to the outer membrane surface; and binding to sites in the membrane fabric. The view that the energized membrane contains high affinity sites for cationic dyes is supported by: comparison of the spectra of the membrane-bound and polyanion-bound dye; competition with divalent cations; osmotic activity; and dependence of the spectral shift on the protein/dye ratio. The data are in accord with the concept that the mitochondriaI membrane acts as a fixed negative-charge system where electrostatic interactions occur in microenvironments of low polarity. [ABSTRACT FROM AUTHOR]

The interactions of sodium dodecylsulfate and hexadecyltrimethylammonium chloride with typtophyl model compounds were investigated by fluorescence, classical difference spectra and thermal perturbation spectra. Oppositely charged models and detergents interact to produce marked spectral changes. Apolar interactions also contribute. The ease of interaction with sodium dodecylsulfate decreases in the order: tryptamine, Lys-Trp-Lys, Leu-Trp-Leu, Gly-Trp-Gly. Glu-Trp-Glu shows little or no interaction. Lys-Trp-Lys forms both soluble and insoluble complexes with sodium dodecylsulfate. [ABSTRACT FROM AUTHOR]

The 13C nuclear magnetic resonance spectra of the membrane-active antibiotic valinomycin and its alkali-ion complexes are interpreted. From the 13C chemical shifts and infrared absorptions of the highly stable K+, Rb+ and Cs+ complexes in methanol it is concluded that the ligand conformations of these complexes must be similar. The essential features are the coordination of all ester carbonyl groups with the cation in addition to the formation of intramolecular hydrogen bonds between the amide groups. In contrast to these results the weaker Na+ complex: of the antibiotic exhibits different spectral properties which are more similar to those of the uncomplexed antibiotic in polar solvents thus indicating the existence of a considerably different ligand conformation in this complex. The structure of the Na+ complex can be characterized by a reduced number of coordinated ester carbonyl groups and intramolecular hydrogen bonds between the amide groups as compared to the complexes with larger cations. The conformational rearrangements occurring during the complexation of cations are compared to those observed for the uncomplexed valinomycin in solvent systems of different polarities. Evidence is presented that the interaction of the uncomplexed antibiotic with polar solvent molecules leads to conformational rearrangements which can be characterized by a stepwise opening of a closed structure stabilized by intramolecular hydrogen bond formation. [ABSTRACT FROM AUTHOR]

The interaction of hydroxymercurybenzene sulfonate (HOHgBzS) with 2-thiouridine (s2Urd) and 2-thiocytidine (s2Cyd) by means of absorption-difference spectroscopy and spectrophotometric titration was investigated. The absorption spectra of the reaction mixtures displayed isosbestic points indicating uniform reactions. Scatchard plots of the titration data revealed the formation of 1:1 complexes with a dissociation constant for s2Urd · HOHgBzS and s2Cyd · HOHgBzS of Kd = 0.55 μM and Kd = 0.54 ???M respectively. The interactions of poly(s2U) and poly(s2C) with hydroxymereurybenzene sulfonate were established by their corresponding absorption difference spectra. The binding of hydroxymercurybenzoato (HOHgBz) to poly(s2C) and poly(s2U) was investigated by means of equilibrium dialysis. The association of HOHgBz to 2-thiocytosine bases in poly(s2C) follows a simple hyperbolic binding isotherm. The Scatchard plot of the binding data disclosed that only a fraction (n = 0.32) of the s2CMP residues in poly(s2C) was accessible to the interaction with HOHgBz. The dissociation constant of this interaction was found to be Kd = 4.3 ???M. The binding of HOHgBz to the helical poly(s2U) is a cooperative process as indicated by the sigmoidal binding cuve. Under saturating conditions all s2UMP residues in poly(s2U) interact with the ligand. [ABSTRACT FROM AUTHOR]

The interaction between sonicated dispersions of purified phospholipids, corresponding to those found in the inner mitochondrial membrane, and the enzymes was studied by kinetic and spectroscopic methods. With glutamate dehydrogenase the charge type of the lipid head group determines the extent of lipid-protein complexing. Phosphatidylcholine did not significantly alter the activity of the enzyme at lipid concentrations up to 1 mM, and the nuclear magnetic resonance (NMR) spectrum of the lipid (l%, w/v) was not altered in the presence of enzyme (up to 2%, w/v). Phosphatidylethanolamine did not affect the activity in the oxidation of glutamate, but interfered with the assay in the reverse direction, possibly owing to Schiff-base formation with 2-oxoghitarate. The negatively charged cardiolipin, which is found uniquely in the inner mitochondrial membrane, strongly and reversibly inhibited the enzyme (50% inhibition with 0.1–20 μM lipid, depending on conditions). Another anionic phospholipid, phosphatidylserine (which does not occur in mitochondrla), also inhibited the enzyme, but less effectively (50% inhibition at about 8 μM). The lipid-enzyme interaction decreases with increasing ionic strength, indicating an electrostatic contribution to the binding. NMR studies suggest that there is also an apolar interaction involving the lipid chains. The studies with malate dehydrogenase concentrated on detecting possible stabilization of this enzyme through interaction with lipids. A clear-cut stabilization was found with lysophnsphatidylcholine. Under conditions such that the enzyme alone lost 50% on the initial activity in about 2 h, lysophosphatidylcholine gave a slight (20%) activation and this activity remained constant for several hours. NMR studies indicated that, as with the related enzyme 3-hydroxybutyrate dehydrogenase, both the polar choline head group and the hydrocarbon chains are involved in the binding of the lipid to the protein. These specific lipid · protein complexes are probably examples of allotopic interactions. This form of complexing may explain aspects of the behaviour of these enzymes in vivo. [ABSTRACT FROM AUTHOR]

Aqueous methanol promotes chlorophyll a aggregation and also that of chlorophyllide a, pheophytin a and pheophorbide a. Comparing the aggregation features of all these molecules leads to the assumption that the phytyl chain of chlorophyll a and its Mg atom play antagonist roles in the association of chlorophyll molecules in polar solvents. [ABSTRACT FROM AUTHOR]

In water-methanol solutions, chlorophyll a forms aggregates absorbing at 745 ± 10 nm or at 710 ± 10 nm. In all cases, chlorophyll also gives a form with no absorption at wavelengths between 350 and 1000 nm, which is considered as an important aggregate of chlorophyll molecules (sieve effect). It is assumed that, in the immediate environment of the chlorophyll a molecules, the water/methanol ratio is the determining factor of the type of aggregate obtained and that water and chlorophyll compete for the “aggregation sites” of chlorophyll. [ABSTRACT FROM AUTHOR]

1. Diethylpyrocarbonate reacts specifically with histidine residues in human ceruloplasmin at pH 6. 2. Carbethoxylation of imidazole groups in ceruloplasmin does not affect the optical absorption in the visible region which is due to paramagnetic Type-1 copper or diamagnetic copper, but has a pronounced effect on electron paramagnetic resonance (EPR) spectra of the protein. The ERP-spectral changes do not involve the Type-1 copper signal or a conversion of diamagnetic copper into paramagnetic copper but indicate a change in the binding situation of Type-2 copper. ERP spectra of the modified protein are pH-dependent over the range pH 5-7. Carbethoxylation of ceruloplasmin and the concomitant ERP--spectral changes parallel a decrease in oxidase activity towards ferrous ions and dimethyl-p-phenylenediamine. Inhibition by diethylpyrocarbonate can be (At least partially) reversed by treatment with hydroxylamine at pH 7, which also restores the ERP spectrum of native ceruloplasmin. [ABSTRACT FROM AUTHOR]

The conformations of 1-anilinonaphthalene-8-sulphonate in 2H2O and C2H3O2H have been determined by proton magnetic resonance spectroscopy, and its fluorescence spectra as a solid, m a methyl-cellulose matrix, and in solution in water, methanol and aqueous MgCl2 have been studied. The aromatic rings of anilinonapbthalene-sulphonate are more nearly coplanar in C2H3O2H than in 2H2O. The NH proton fails to exchange with 2H from the solvent in C2H3O2H, and its chemical shift indicates that there is a strong interaction with the sulphonate group. The solvents in which rapid exchange of the NH proton occurs are those in which the quantum yield of fluorescence of anilinonaphthalene-sulphonate is quenched. The fluorescence emission is enhanced and blue-shifted in strong aqueous MgCl2 solutions. Under some conditions the overall emission is composed of contributions from two separable spectra. It is argued that an efficient mechanism for quenching the first excited singlet of anilinonaphthalene-sulphonate is through vibrations of bonds to the nitrogen, and that the shifts of emission maxima in non-aqueous solvents may be partly a result of the molecule displaying different spectra m different conformations (rather than simply a solvent-polarity-induced change). There is no convincing reason for the assumption that when a fluorescence enhancement follows the binding of anilinonaphthalene-sulphonate to a biological structure the site of interaction must be hydrophobic. [ABSTRACT FROM AUTHOR]

Angiotensinamide II has been studied by circular-dichroism spectroscopy in comparison with some of its peptidic fragments and structural analogs. The following observations have been made. The stepwise examination of elongated peptides shows the appearance of a structural organisation at the heptapeptide stage. Evidence that C-terminal phenylalanine residue plays an important role on the overall conformation of the octapeptide is obtained. As already suggested for some other short peptides, circular-dichroism spectra of angiotensinamide II show dramatic perturbations of the peptidic region due to side-chain aromatic-chromophore overlapping. The contribution of prolyl residue in position 7 of the peptide appears to be critical; equilibrium between cis and trans forms is believed to be shifted toward one or the other conformer upon binding to receptor site. This would result in the adequate positioning of the phenylalanine residue, the biological importance of which has already been shown. [ABSTRACT FROM AUTHOR]

The exchange of amide hydrogens of pork and beef insulin with solvent deuterium has been followed for 120 rain by infrared spectroscopy. Solutions made by dissolving carefully dried insulin in dilute ²HCl were maintained at 40 °C, and pH of 2.35 throughout the period of observation. The number of unexchanged amide hydrogens at a given time has been calculated from the absorbance at 1541 cm-1. A computer programme has been used to calculate the best two-term exponential to fit these data. This gives the number of hydrogens and their rate constant for exchange for each of two different groups. The number of hydrogens which exchanged too fast to be observed has been calculated from the difference between the total number of amide hydrogens (48) and the sum of those found from the two-term exponential. Pork insulin appears to have 9 amide hydrogens in the slowest exchanging group, 14 or 15 in the intermediate group and 25 or 24 in the fast group. Beef insulin appears to have 8 or 9 in the slowest group and their rate of exchange is higher than for the corresponding group in pork insulin, 11-13 in the intermediate group and 29-26 in the fast group. Attempts have been made to assign each amide hydrogen of pork insulin to a particular group. [ABSTRACT FROM AUTHOR]

1. Nuclear-magnetic-resonance spectroscopy was used to follow the course of the urocanase reaction. It was shown that 3-(4'-hydroxyimidazol-5'-yl)-propionate is the true enzyme product and that it spontaneously tautomerises to racemic 3-(imidazol-4'-one-5'-yl)-propionate. 2. Conversion of urocanate in deuterium oxide by a crude-enzyme preparation from Pseudomonas putida led to a product containing two adjacent CH²H-groups. Oxidative degradation of this product afforded optically pure (RR)-[²H2]succinic acid. This fining defines the stereochemistry of proton addition to the double bond of urocanate. [ABSTRACT FROM AUTHOR]

D-Glyeeraldehyde-3-phosphate dehydrogenase has been reacted with spin labels, which were designed to elucidate the geometry of the active site of the enzyme. Three series of spin labels were used, which represented either substrate and coenzyme analogs or irreversible inhibitors, the separation of the active site directing part from the nitroxide radical being readily adjusted through the length of the intervening methylene chain. The effect of this variation on the electron paramagnetic resonance spectrum of the labeled protein as well as the effect, of coenzyme binding on these spectra have been investigated. All spectra are complex and represent the superposition of at least three different types of spectra. These three types of spectra are correlated to nitroxide radicals in three different types of motion: (a) very fast rotation in a fluid region, (b) medium fast rotation of a hindered radical and (c) slow rotation of an almost immobilized radical. The spin labels differ only in the relative population in the three states related to the three types of motion. Also addition of the coenzyme NAD only alters this relative population. A spin labeled coenzyme analog, which has the nitroxide in the region of the sugar-nicotinamide bond, is bound to the protein and displays an electron paramagnetic resonance spectrum typical for an immobilized radical. The geometry of the active of D-glyceraldehyde-3-phosphate dehydrogenase is complex and cannot be described as a simple cleft, but instead as a system of narrow and wider spaces, which is changed by the binding of the coenzyme. [ABSTRACT FROM AUTHOR]

X-ray small angle scattering was performed on solutions of the bacteriophages fr and R17. The scattering curves of the phages are nearly identical. The radius of gyration was found to be 105.2 Å, the molecular weight as obtained from a high precision measurement is 3.62 × 106. The radial excess electron density distribution, derived from measurements in 0.02 M Tris-Cl buffer pH 7.2 confabbing 0.15 M NaCl and 1 mM EDTA, indicates that the phages have a mean outer radius of 131.7 Å and consist of a shell and a less dense core. The core consists of RNA as is shown by measurements in sucrose solutions. It contains only about 80% of the RNA, the remaining 20% are considered to penetrate into the shell. Models calculated assuming the mean outer radius of the RNA to be 107.8 Å yield a mean inner radius of the protein shell of 104.0 Å. The amount of water associated with the phages is found to be about 0.9 g/g. [ABSTRACT FROM AUTHOR]

The interaction of concanavalin A, the phytohemagglutinin of the jack bean, with a variety of glycosides has been studied by the technique of ultraviolet difference spectroscopy. Whereas methyl α-D-gluco- and manno-pyranoside gave rise to relatively low intensity difference spectra, p-nitrophenyl α-D-mannopyranoside and α-D-glucopyranoside yielded large difference spectra upon interaction with concanavalin A. Suing this technique as a measure of concanavalin A activity, ii was demonstrated that the protein specifically binds low molecular weight carbohydrates at much lower pH values (e. g. pH 2.4) than previously believed. Although polysaccharides arc also bound at these low pH values, they are not precipitated by concanavalin A. Molecular weight studies in acid media indicate that the protein does not dissociate and it is suggested that electrostatic repulsion of the protein molecules due to their high net positive charge prevents protein-polysaccharide lattice formation and hence failure of the complex to precipitate. [ABSTRACT FROM AUTHOR]

The circular dichroism of sixteen DNA samples from fourteen species has been determined. Plots of molar ellipticity against base composition show considerable scatter, which is much too great to be accounted for in terms of reproducibility of materials and measurements. The data are most simply fitted with the assumption of linearity; extrapolation then leads to apparent, circular dichroism curves for 0 and 100% (A+T). These are regarded as having probably no physical reality, but serve as standards for the calculation of circular dichroism curves for DNA of any given composition. For the materials used in the present work the discrepancy between the observed curves and those calculated from the formal standards is at worst not more than about 10%, and much better than this over most of the range, particularly on the long-wavelength side of the crossover. The use of a quadratic function, based on the assumption that the circular dichroism can be operationally expressed, like hypochromicity, in terms of three spectroscopic parameters, offers no significant advantage in describing the circular dichroism data. [ABSTRACT FROM AUTHOR]

High resolution nuclear magnetic resonance spectroscopy is used to study conformational changes in histone fractions F2a1 and Fi when the ionic strength of aqueous solutions is raised. Increasing line widths of certain resonance peaks, in particular those of apolar and aromatic amino acids, together with sequence data lead to the conclusion that the C-terminal half of F2a1 and a central portion of Fi are revolved m the conformational changes. The proportion of amino acid residues incorporated into secondary structure (as indicated by optical rotatory dispersion) less than that involved in the conformational changes indicated by the nuclear magnetic resonance results. Intermolecular interactions are therefore postulated to explain this difference and these are specific in as much as they involve only a part of the histone molecule and include the regions of the chain having high potential for secondary structure formation. [ABSTRACT FROM AUTHOR]

Proton magnetic resonance spectra are reported of α-lactalbumin at 100 and 220 MHz. The extensive sequence homologies between this protein and lysozsme have led to the postulate of a close conformational similarity. Our results on optical rotatory dispersion, circular dichroism and infrared spectra of the two proteins are consistent with this hypothesis. The proton magnetic resonance spectra of the native proteins show differences in the upfield region of the spectrum associated with aliphatic side chain protons. Calculations of ring-current shifts induced in these protons, in terms of the atomic coordinates for the putative α-lactalbumin structure, as fitted by Phillips and co-workers to the lysozyme model, account semi-quantitatively for the observed spectrum. The discrepancies between the α-lactalbumin and lysozyme spectra are, moreover, almost wholly explicable in terms of specific sequence differences between the two proteins. [ABSTRACT FROM AUTHOR]

The pig liver glutamic dehydrogenase has been studied by difference spectroscopy and shows two diphosphopyridine nucleotide binding sites per protomer of molecular weight 52 000. A first NADH molecule binds on an adenylic site (Ka = 4.2 mM-1), and a second molecule on an nico- tinamide site (Kn = 13 mM-1). NADPH binds only on the nicotinamide site (K = 21 mM-1). The adenylic site does not only strongly bind ADP (K = 2800 mM-1), but also ADP-ribose, ATP, AMP, NADH, NAD, ApA, GpA, with a decreasing affinity; ApG and GTP does brad on tiffs site which is specific of the adenosine 5'-phosphate group. The high ionic strength decreases ADP binding. The linkage between the nicotinamide site and NADH induces a positive Cotton effect in the range of the absorption band of the reduced nicotinamide group; moreover the absorption spectra of bound NADH is shifted about 5 nm to tho greater wavelengths with an hypochromism of 6%, with respect to free NADH. The evidence for two distinct binding sites is given by: (a) the existence of two difference spectra: the first due to the nicotinamide chromophore perturbation and the second due to the adenylic chromophore perturbation. This last is sitar to that obtained with ADP, AMP, ATP; etc.; (b) the competition between ADP and NADH only on the adenylic site; (c) the fact that the saturation curves obtained from measurements made in the absorption range of adenosine and reduced nicotinamide chromophores are different and square with the theoretical saturation curves of two distinct sites per protomer. NAD also binds on the adenylic site in competition witch ADP, whereas NADP and NADPH do not induce any perturbation of the adenosine chromophore. All these results are in good agreement with the interpretations given by Frieden to explain the effects observed in the steady-state enzymatic kinetics at high concentrations of coenzyme. [ABSTRACT FROM AUTHOR]

The solubilization of the polycyclic hydrocarbon benzo[a]pyrene by aqueous solutions of dAMP-Ag+ complexes has been investigated. At high pH solubilization is efficient at Ag+/dAMP ratios not more than 1 but is markedly diminished when the ratio of total silver ions added per base residue is greater than 2. The components separately show little solubilizing activity. The ultraviolet absorption and circular dichroism spectra of shyer complexes of dAMP and poly A under various conditions have been compared. [ABSTRACT FROM AUTHOR]

Abstract It is shown that when a deterministic signal is observed in quasi-linear noise (herein defined), the usual estimate of the signal amplitude spectrum for a finite time interval converges uniformly and exponentially fast in probability to this spectrum as the length of the time interval becomes infinite. When the limiting amplitude spectrum exists, the estimate tends to this function exponentially fast but no longer uniformly,in general. [ABSTRACT FROM AUTHOR]

The infrared spectra of the four deoxynucleosides dT, dA, dG, and dC have been studied in ²H2O solution without and with addition of CuSO4. The infrared spectra of dT and dA remain unchanged by addition of copper(II), whereas new bands appear at 1,517 and 1,547 cm-1 in the infrared spectrum of dC and at 1,590 cm-1 in the infrared spectrum of dG after addition of CuSO4. The changes in the infrared spectra of DNA from Streptomyces chrysomallus (72 mole% guanine + cytosine) after interaction with copper(II) are very similar to the observed changes in the nucleoside spectra. New bands at 1,518 and 1,551 cm-1 indicate the interaction of cytosine bases of DNA with copper(II), the band at 1,590 cm-1 indicates the interaction of guanine bases of DNA with copper(II). [ABSTRACT FROM AUTHOR]