Ultraviolet Difference Absorption Research Articles

Effect of ultrasound on mill starch and protein in ultrasound-assisted laboratory-scale corn wet-milling

Ultrasound was applied to facilitate starch-protein separation during ultrasound-assisted laboratory-scale corn wet-milling. The effects of ultrasound on the properties of mill starch and protein were investigated to explore the possible mechanisms in this study. A higher starch yield was obtained with sonication treatment in starch-protein separation (15 min, 200 W) and the results showed that ultrasound did not induce damage to the product starch. The differences in the distribution of water proton transverse relaxation time for mill starch suggested that the interaction between starch granules and protein in mill starch was destroyed. The free sulfhydryl content increased by 37.21% and the disulfide bond content decreased by 43.66% of the separated proteins indicated that ultrasound destroyed the disulfide bonds effectively (p < 0.05). Sonication treatment for longer time or at higher power decreased α-helix and β-turn while increased β-sheet and random coil (p < 0.05). Ultrasound increased surface hydrophobicity and decreased the thermal stability of the protein (p < 0.05). The higher intensity of ultraviolet–visible spectroscopy and ultraviolet-difference absorption spectroscopy indicated the unfolding of protein by sonication treatments. Ultrasound may break down the disulfide bonds and hydrogen bonds of protein molecules, effectively releasing starch from the disintegrated protein agglomerates.

Regulation of Tissue Factor Activity: Conformational Changes in Tissue Factor Associated with Cleavage of the Cysteine 186-Cysteine 209 Disulfide Bond.

Tissue factor (TF) exists in an encrypted, inactive form and an active, procoagulant form. The extracellular domain of TF contains two disulfide bonds. The crystal structure of the active form of TF reveals Cys186 and Cys209 in an unusual solvent-exposed disulfide bond. It was recently proposed that encrypted TF contains two free thiols at Cys186 and Cys209, and formation of a disulfide bond between Cys186 and Cys209 is associated with the decryption of TF (Chen et al, Biochemistry 2006). To characterize the structural differences between the putative encrypted TF and active TF, containing one and two disulfide bonds respectively, we developed an assay to quantitate the free thiols and disulfide bonds in recombinant TF and examined the solvent accessibility of the two disulfide bonds under native and denatured conditions. The perturbations of chromophores of TF by UV difference spectroscopy during specific reduction of Cys186–Cys209 were then examined. In order to quantitate the free thiols and disulfide bonds, TF was subjected to Alexa 488-maleimide (green) labeling to identify any free thiols and, after reduction with TCEP, a second labeling with Alexa 647-maleimide (red) to identify newly exposed free thiols formerly involved in disulfide bonds. Alexa-labeled TF was subjected to SDS gel electrophoresis and the gel was developed on a fluorescence imager to provide quantitative analysis of the integrated fluorescence intensity for each Alexa dye separately. The molar concentrations of free thiol in TF before TCEP treatment and thiols exposed after reduction were determined by comparison to calibration curves that were generated using Alexa maleimide-albumin conjugates. Recombinant TF in solution contains 0.2 moles of free thiol. Upon treatment with 10 mM TCEP under native conditions, TF is partially reduced and contains 1.97 moles of half cystine thiols. TF is fully reduced by 10 mM TCEP under denaturing conditions in the presence of 4 M urea (4.29 moles of thiols, theoretical=4.0 moles). This is consistent with the observation that only one disulfide bond is solvent-exposed in native TF and is accessible for reduction. We then performed ultraviolet absorption difference spectroscopy using the partitioned cell technique to monitor any structural transition during partial reduction of TF. The UV difference spectrum comparing the TCEP-treated TF and unreduced TF showed three peaks, one at 278 nm, a major peak at 285 nm and a small shoulder at 292 nm, that is characteristic of red-shifts in absorption of both tyrosine and tryptophan. Under the conditions employed, this conformational transition was completed in 60 minutes. These results comparing fully oxidized, active TF and partially reduced, encrypted TF indicate that tyrosine and tryptophan residues in TF become protected from solvent upon reduction of the Cys186–Cys209 disulfide bond. In conclusion, we have demonstrated by a new assay that only one disulfide bond within active TF is solvent-exposed and accessible for reduction. The partial reduction of TF at this disulfide bond results in a conformational transition that is associated with the protection of tyrosine and tryptophan residues, indicating structural difference between active TF and encrypted, inactive TF.

The unfolding intermediate state of calf intestinal alkaline phosphatase during denaturation in guanidine solutions.

The equilibrium unfolding of calf intestinal alkaline phosphatase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and ultraviolet difference spectra. At low concentrations of GdmCl (< 1.6 M), the fluorescence intensity decreased with a slight red shift of the emission maximum from 332 nm to 344 nm. An unfolding intermediate state was observed at a broad concentration range of GdmCl as a denaturant (between 1.6 and 2.6 M). This intermediate was characterized by increased fluorescence emission intensity, ultraviolet difference absorption at 236 nm and 260 nm, as well as increased binding to the protein and red shift of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid.

Interaction of nucleosome core DNA with transition proteins 1 and 3 from boar late spermatid nuclei.

The DNA binding properties of boar transition protein 1 and 3 (TP1 and TP3) were studied by means of physicochemical techniques. The ultraviolet difference absorption spectra upon TP1 and TP3 binding to rat liver nucleosome core DNA (double-stranded DNA) showed TP1- and TP3-induced hyperchromicity at 260 nm, which is suggestive of local melting of DNA. CD measurements of TP1-DNA and TP3-DNA complexes indicated that the binding of TP1 and TP3 induced different conformational changes in DNA, probably including local melting of DNA. Thermal melting studies on the binding of TP1 and TP3 to DNA showed that although at 1 mM NaCl TP1 and TP3 caused slight stabilization of the DNA against thermal melting, destabilization of the DNA was observed at 50 mM NaCl. From the results of quenching of the tyrosine fluorescence of TP1 and the tryptophan fluorescence of TP3 upon their binding to double-stranded and single-stranded boar liver nucleosome core DNA at 50 mM NaCl, the apparent association constants for the binding of TP1 to double- and single-stranded DNA were calculated to be 8.0 x 10(4) and 1.3 x 10(5) M-1, respectively, and those for the binding of TP3 to double- and single-stranded DNA to be 7.1 x 10(4) and 1.8 x 10(5) M-1, respectively. These results suggest that TP1 and TP3, having higher affinity for single-stranded DNA, induce local destabilization of DNA, probably through the stacking of Tyr32 and Trp18 with nucleic acid bases, respectively.

Comparison of inactivation and conformational changes of aminoacylase during guanidinium chloride denaturation

The inactivation and unfolding of aminoacylase (EC 3.5.1.14) during denaturation by different concentrations of guanidinium chloride (GuHCl) have been compared. A marked decrease in enzyme activity is already evident at low GuHCl concentrations before significant unfolding of the enzyme molecule, as monitored by fluorescence, ultraviolet difference absorption and CD measurement. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of aminoacylase during denaturation by GuHCl. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction kinetics were found to be a monophasic first-order reaction. The kinetics of the unfolding were a bisphasic process consisting of two first-order reactions. At lower GuHCl concentration (< 1.0 M), the enzyme activity was stripped at a high rate whereas its conformation was only slightly affected. At 1.0 M GuHCl, the inactivation rate of the enzyme was much faster than the unfolding rate. At higher GuHCl concentrations (> 1.0 M), the inactivation rate was too fast to be measured by conventional dynamic methods, whereas the unfolding remained as a bisphasic process with the fast reaction accruing very fast and the slow reaction occurring at a measurable rate. The results suggest that active sites of aminoacylase containing Zn 2+ ions are situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.

Identification of amino acid residues responsible for the changes of absorption and fluorescence spectra on the binding of subtilisin BPN' and Streptomyces subtilisin inhibitor.

An ultraviolet absorption difference spectrum characteristic of the ionization change of a tyrosyl residue was observed on the binding of subtilisin BPN' with Streptomyces subtilisin inhibitor (SSI) at alkaline pH. This difference spectrum was considered to be induced by a pKa shift (from 9.7 to > or = 11.5) of a tyrosyl residue of subtilisin BPN' in the interaction with carboxyls of SSI [Inouye et al. (1979) J. Biochem. 85, 1115-1126]. In the present paper, the tyrosyl residue in subtilisin BPN' and the carboxyls in SSI were identified by analyzing the difference spectrum using mutants of subtilisin BPN' and SSI: naturally occurring mutants and those prepared by site-directed and cassette mutagenesis. The difference spectrum disappeared on the binding of a mutant subtilisin BPN' of which Tyr104 was replaced by Phe (S-BPN'Y104F) and SSI at pH 9.8. The magnitude of the absorption difference was much smaller when subtilisin BPN' was bound with a mutant SSI of which both Glu67 and Asp68 were replaced by Gly than with the wild-type SSI. These lines of evidence indicated that the difference spectrum was caused by Tyr104 of subtilisin BPN' interacting with Glu67 and Asp68 of SSI. The binding of subtilisin BPN' and SSI is accompanied by an increase of tryptophan fluorescence, which is pH-dependent in the range of pH 7-11 [Uehara et al. (1978) J. Biochem. 84, 1195-1202]. In the present study, this pH-dependence of the fluorescence diminished when SSI bound with S-BPN'Y104F. This suggested that the fluorescence increase was due to Trp106 of subtilisin BPN' and was influenced by the ionization of Tyr104.

Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phospholipase A 2 with micellar lipid/water interfaces of competitive inhibitors

In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249–257 and references therein) we have demonstrated that synthetic ( R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A 2 (PLA 2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA 2 and of an inactive PLA 2 in which the active site residue His-48 has been modified by alkylation with l-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the ( R-configuration in its active site, whereas the ( S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the ( R)-isomer than those of the ( S)-isomer. The modified PLA 2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the ( R) or the ( S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitory phospholipid analogue, the progressive binding of a single ( R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the large difference in overall dissociation constants of the complexes between enzyme and micelles of ( R)-and ( S)-inhibitors. With the use of PLA 2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.

Tryptophan residues of phospholipase A 2 from the venom of an Australian elapid snake (Pseudechis australis)

S. Nishida and N. Tamiya. Tryptophan residues of phospholipase A 2 from the venom of an Australian elapid snake ( Pseudechis australis). Toxicon 29, 429–439, 1991.—Tryptophan residues 31 and 69 (Trp-31 and Trp-69) in phospholipase A 2 (Pa-11) from the venom of an Australian elapid snake, Pseudechis australis, were modified with N-bromosuccinimide (NBS) or with 2-nitrophenylsulphenylchloride (NPSC1). NBS oxidized only Trp-31, whereas NPSC1 reacted with both Trp-31 and Trp-69. Treatment of the enzyme with NBS at various pH values resulted in losses of enzymic and lethal activities. No protective effect on the oxidation with NBS was observed by the addition of calcium ion (20 mM) or lecithin (4 mM). The observations suggest that Trp-31 is exposed to the surface of the molecule, composes a part of the lipid-water interface recognition site around the active site and is essential for enzymic activity. Calcium ion addition to the solution caused a change in ultraviolet spectrum of the native enzyme Pa-11. The difference spectrum indicates that a charge effect caused a typical tryptophan blue shift in the Ca 2+-enzyme complex. Pa-11 oxidized with NBS showed a smaller ultraviolet absorption difference on the addition of Ca 2+ ion. The results show that the hypochromic effect induced upon the binding of Ca 2+ is due to perturbation of the specific tryptophan residue (Trp-31) which is involved in the active site. Dissociation constant, Kd, of the Ca 2+-enzyme complex was calculated to be 3.4 × 10 −4 M at pH 8.0.

Photophysics of metalloazurins

The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(II) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(II) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(II) azurin Pae were measured between 30 and 160 K and showed little change in value. Ultraviolet absorption difference spectra between metalloazurin Pae and apoazurin Pae were measured, as were the fluorescence spectra of metalloazurins. These spectra were used to determine the spectral overlap integral required for dipole-dipole resonance calculations. All metalloazurins exhibit a reduced fluorescence lifetime compared to their respective apoazurins. Forster electronic energy transfer rates were calculated for both metalloazurin Pae and metalloazurin Afe derivatives; both enzymes contain a single tryptophyl residue which is located in a different position in the two azurins. These azurins have markedly different fluorescence spectra, and electronic energy transfers occur from these two tryptophyl sites with different distances and orientations and spectral overlap integral values. Intramolecular distances and orientations were derived from an X-ray crystallographic structure and a molecular dynamic simulation of the homologous azurin Ade from Alcaligenes denitrificans, which contains both tryptophyl sites. Assignments were made of metal-ligand-field electronic transitions and of transition dipole moments and directions for tryptophyl residues, which accounted for the observed fluorescence quenching of Hg(II), Co(II), and Ni(II) azurin Pae and Cu(II) and Hg(II) azurin Afe. The fluorescence of azurin Pae is assigned as a 1Lb electronic transition, while that of azurin Afe is 1La. The marked fluorescence quenching of Cu(II) azurin Pae and Cu(I) azurin Pae and Afe is less well reproduced by our calculations, and long-range oxidative and reductive electron transfer, respectively, are proposed as additional quenching mechanisms. This study illustrates the application of Forster electronic energy transfer calculations to intramolecular transfers in structurally well characterized molecular systems and demonstrates its ability to predict observed fluorescence quenching rates when the necessary extensive structural, electronic transition assignment, and spectroscopic data are available. The agreement between Forster calculations and quenching rates derived from fluorescence lifetime measurements suggests there are limited changes in conformation between crystal structure and solution structures, with the exception of the tryptophyl residue of azurin Afe, where a conformation derived from a molecular simulation in water was necessary rather than that found in the crystal structure.

Electrophoretic and spectroscopic analyses of equine α 2-macroglobulin with cleavage of the thiol ester bonds by methylamine

Reaction of equine α 2-macroglobulin (α 2M) with methylamine caused generation of 3.7 mol of thiol groups per mole of the protein, and the second-order rate constant of the generation was calculated to be 3.5 m −1 s −1. The inhibitory profile of caseinolytic activity of trypsin indicated that one molecule of equine α 2M inhibited two molecules of trypsin, similar to human α 2M. The methylamine-treated equine α 2M, with complete cleavage of the thiol ester bonds, still inhibited the activity of trypsin, though human α 2M lost its inhibitory activity by treatment with methylamine. These results indicate that the mode of inhibition of trypsin by equine α 2M is substantially unperturbed by cleavage of the thiol ester bonds and that the intact thiol ester bonds per se are not essential for the ability of equine α 2M to bind the enzyme. Ultraviolet absorption difference, intrinsic fluorescence, and circular dichroism spectra of the methylamine-treated equine α 2M showed that this treatment caused only a small change in conformation of the protein. Reaction of the methylamine-treated protein with trypsin induced appreciable changes in the spectra, indicating a large change in conformation of the protein. These findings were consistent with the results obtained by electrophoresis: The band of methylamine-treated equine α 2M showed indistinguishable mobility from that of the unmodified protein, indicating that no appreciable change in conformation occurred, and distinctly different mobility from that of the unmodified or methylamine-treated equine α 2M when each had reacted with trypsin.

Assessment of the α 1β 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (α 2 +β 2 and α 2β 2 +: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the α 1β 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both α 2 +β 2 and α 2β 2 + showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the α 1β 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.

Essential tryptophan residues in the function of cellulase from Schizophyllum commune

The tryptophan residues of the cellulase (EC 3.2.1.4; 1,4-β- d-glucan 4-glucanohydrolase) from Schizophyllum commune were oxidized by N-bromosuccinimide in both the presence and absence of substrates and inhibitors of the enzyme. In the absence of protective ligands, eight of the twelve tryptophan residues in the cellulase were susceptible to modification with concomitant inactivation of the enzyme. The binding of the substrates, CM-cellulose, methyl cellulose, cellohexaose or lichenan and the competitive inhibitor, cellobiose, protected one tryptophan residue from oxidation but did not prevent the inactivation. Characterization of the oxidized enzyme derivatives by ultraviolet difference absorption and by fluorescence spectroscopy indicated that two tryptophan residues are essential in the mechanism of cellulase catalysis. One residue appears to be directly involved in the binding of substrate, while the second residue is proposed to constitute an integral part of a catalytically sound active centre.

Hyperchromic effect of collagen induced by human collagenase.

Loss of the highly ordered triple-helix structure of native collagen on denaturation or enzymatic degradation involves a helix-to-coil transition, which can be seen as an increase at 227 nm in its ultraviolet difference absorption spectrum. We report here the successful use of this hyperchromic effect to quantify collagen in solution and to follow up the time-course of collagen degradation catalyzed by collagenase. Using 14C-labelled collagen substrate we show the excellent correlation between enzyme-induced increase in ultraviolet difference absorption and formation of specific cleavage products. The novel method was found to be suitable to characterize the enzymatic properties of human leukocyte collagenase. Activation of latent collagenase to the active enzyme could be followed continuously and an activation lag estimated.

Difference spectrophotometric study on the interaction of cellulase from Schizophyllum commune with substrate and inhibitors

Studies were made on the ultraviolet difference absorption spectra of cellulase (1,4-β- d-glucan 4-glucanohydrolase, EC 3.2.1.4) from Schizophyllum commune specifically induced by substrates and inhibitors. The substrate, cellodextrin, and the competitive inhibitor, cellobiose, produced characteristic difference spectra with maxima at 291–292 nm, 283–286 nm and 278–280 nm. Such difference spectra indicate that the environment of a tryptophan residue or residues located near to or at the active site of S. commune cellulase becomes more hydrophobic upon the binding of these ligands. The spectrum produced by another competitive inhibitor, glucose, with a broad maximum at 278–283 nm and a shoulder at 290 nm, was similar to the simple solvent perturbation spectrum induced by ethylene glycol. The differences between the cellulase spectra induced by these ligands suggest a subsite structure for the binding of substrate/inhibitor to the enzyme, with cellobiose and cellodextrins, but not glucose, binding to a subsite that contains the tryptophan residue or residues. From the concentration dependence of the difference spectra, the dissociation constants for the cellulase complex with cellobiose and cellodextrin were estimated to be 22 mM and 3.5 mM, respectively. The Δϵ max value at 291–292 nm for the cellulase-cellobiose complex was calculated to be 1710 M −1 · cm −1, suggesting that one tryptophan residue near or at the active site is specifically perturbed by cellobiose. In contrast, the cellulase-cellodextrin complex provided a Δϵ max value at 291–292 nm of 2610 M −1 · cm −1, indicating that approximately two tryptophan residues are specifically perturbed by this substrate. The numbers of tryptophan and tyrosine residues which were exposed and accessible to dioxane were determined by the solvent perturbation technique to be eight each. In the presence of either cellobiose or cellodextrin, two tryptophan residues and one tyrosine residue were masked from dioxane perturbation.

Temperature-induced ultraviolet difference absorption spectrometry for determination of enthalpy changes: Binding of 4-methylumbelliferyl glycosides to four lectins

Raising the temperature in a single mixture of a lectin and a chromophoric glycoside allows determination of the binding enthalpy. This is made possible by continuously monitoring the displacement of the complex from its equilibrium concentration with a sensitive difference absorption spectrophotometer. The method is illustrated with the following lectins: concanavalin A, soybean agglutinin, peanut agglutinin and Erythrina cristagalli agglutinin. The ligands are 4-methylumbelliferyl glycosides. The binding enthalpies found range from −60 kJ · mol −1 for the Galß1 → 3GalNAc-ßglycoside and peanut agglutinin to −30 kJ · mol −1 for a monosaccharide glycoside and the other lectins.

Interaction of cholera toxin with gangliosides: differential effects of the oligosaccharide of ganglioside GM1 and of micellar gangliosides

Ultraviolet difference absorption spectra of cholera toxin and its B protomer produced by the oligosaccharide moiety of the monosialoganglioside GM1 were measured as a function of the oligosaccharide concentration. In the presence of oligosaccharide, the spectrum is characterized by three peaks at 282, 288, and 292 nm. A linear increase in difference absorption was observed at these wavelengths vs. oligosaccharide concentration; a saturation effect occurred when the molar ratio of oligosaccharide to cholera toxin was higher than 5. The features of the spectra indicated that the binding with the oligosaccharide affected the environment of tryptophan and tyrosine residues of protomer B. In good agreement with the above results, circular dichroic spectra indicated also a local effect of the binding, mostly restricted to protomer B, while the residues of protomer A remained largely unperturbed. Difference absorption spectra were also measured for cholera toxin in the presence of ganglioside and detergent micelles. The employed gangliosides GD1a and GT1b, unable to bind cholera toxin, interact with the protein by way of contaminating traces of GM1. The preparations of GD1a and GT1b contained 0.8-1.0% (w/w) and 0.4-0.5% (w/w) of GM1, respectively. The results obtained with ganglioside GD1a and GT1b in contrast with the observations made with the oligosaccharide of GM1 indicated a major conformational change of the toxin structure. Upon comparison of the conformational change induced by ganglioside micelles with that induced by sodium dodecyl sulfate it may be suggested that the ganglioside micelle, behaving as a detergent, alters the structure of the toxin such as to induce the penetration of protomer A into the lipid milieu.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of D-phenylalanine with Co(II)-substituted rabbit muscle pyruvate kinase: kinetic and optical properties.

The kinetic and optical properties of Co(II)-substituted pyruvate kinase in the presence of D-phenylalanine (D-Phe) were investigated. The results are discussed in comparison with the effects of its optical isomer L-phenylalanine (L-Phe) on the same enzyme. The catalytic effect of D-Phe on rabbit muscle pyruvate kinase depended upon the nature of the activating divalent metal ion used. It has stimulatory effect on Mg(II)-activated enzyme, but inhibitory effect on Co(II)-activated enzyme. Unlike the inhibitory effect of L-Phe, the inhibition of Co(II)-enzyme by D-Phe was not sensitive to the changes of pH and temperature, could not be reversed by L-alanine (L-Ala), displayed hyperbolic kinetics, and was noncompetitive with respect to phosphoenopyruvate saturation. D-Phe induced substantial visible circular dichroism (CD) spectral changes of Co(II)-enzyme similar to those induced by L-Phe. Although ultraviolet CD spectrum was not affected, D-Phe induced an ultraviolet difference absorption spectral change very similar to, but much smaller than, that induced by L-Phe. Our results support that D-Phe and other amino acids interact with the enzyme at two different sites: a common site, causing similar conformational changes which bear little direct kinetic relevance, and a kinetically relevant site, which is sterically dependent upon the side chain of the amino acids.

Binding of porcine pancreatic phospholipase A2 to various micellar substrate analogues. Involvement of histidine-48 and aspartic acid-49 in the binding process.

The interaction of porcine pancreatic phospholipase A2 (PA2) with micelles of various single-chain phospholipid analogues was studied by ultraviolet absorption difference spectroscopy and light-scattering measurements. The phospholipids used were either substrate analogues or products, varying in hydrocarbon chain lengths and polar head groups. The results indicate that the enzyme forms a stable complex over a wide range of enzyme and lipid concentrations. From the equivalent "molecular weight" and from the lipid to enzyme molar ratio (N) of the micelle--enzyme complex, it can be calculated that complexes containing saturated hydrocarbon chain lipids generally consist of two enzyme molecules and half of the number of lipid monomers present in free micelles. The interaction forces between the enzyme and lipid monomers bound in the complex are mainly hydrophobic. Stronger binding is found when the essential cofactor Ca2+ is bound to the enzyme. pH-titration studies on the binding of native PA2 to aggregated lipid structures showed that at least one group with a pKA value of 6.25 is involved in the interaction with lipid micelles. At acidic pH, micelle binding is stronger than at neutral or alkaline pH. Alkylation of the active site residue His48 resulted in a shift of the pKA value to 4.6, while addition of Ca2+ appears to stabilize the micelle-binding conformation of both native and modified enzymes over a broad pH range (pH 4--9.5). From these observations it is suggested that both the Ca2+ binding residue Asp49 [Fleer, E. A. M., Verheij, H. M., & de Haas, G. H. (1980) Eur. J. Biochem. 113, 283--288] and His48 control micelle binding of the native enzyme. For optimal binding in the absence of Ca2+, a long-distance hydrogen bond between these two residues is required; this can be established via a water molecule. It is assumed that it is a proton of this "H bond" which is titrated with a pKA value of 6.25. When the "H bond" is absent, as in the alkylated enzymes, Asp49 alone controls micelle binding with a pKA of 4.6. These results, together with the effect of Ca2+ on micelle binding, indicate that it is not the "hydrogen bridge" between His48 and Asp49 which is of main importance for an optimum binding conformation of the enzyme but the effective charge in the microenvironment of Asp49. It is proposed that a negative charge on this carboxylate causes a conformational change of the enzyme which leads to a protein conformation lacking an active micelle binding site. Binding of Ca2+ or reprotonation neutralizes this negative charge and restores the enzyme's ability to bind micelles.

Triplet state properties of the methylmercury(II)-tyrosine complex

CH 3Hg(II)OH forms complexes at pH 8 with tyrosine and with tyrosine ethyl ester (TEE) that are detected by ultraviolet difference absorption spectra. With K f defined by CH 3HgOH + HB ▪ CH 3HgB + H 2O, we find log K f = 3.61 (tyrosine) and 3.36 (TEE). A heavy-atom effect is observed in frozen glasses of the complexes; this indicates a close interaction between Hg and the chromophore. No UV difference spectrum or heavy-atom effect is observed with N-acetyl tyrosine ethyl ester, indicating that complexing at the phenol O does not occur, and suggesting that binding occurs at the amine N. Zero field optically detected magnetic resonance (ODMR) measurements of the CH 3Hg(II)-tyrosine triplet state give (D, E) = (0.129, 0.047) or (0.134, 0.041) cm −1 depending upon assignment of transitions. D of tyrosine is relatively unaffected, but E is reduced by CH 3Hg(II) complexing. Low-temperature kinetic measurements show that the shortest lived sublevel of the complex is T z , where z lies along the phenol long axis in tyrosine. A dominant 11.6-msec component in the 77 K decay of the phosphorescence is consistent with the individual sublevel lifetimes obtained by ODMR.

The hydrodynamic shape, conformation, and molecular model of Escherichia coli ribosomal 5 S RNA.

The structure of ribosomal 5 S RNA has been examined using several physical biochemical techniques. Hydrodynamic measurements yield a s020,omega and [eta] of 5.5 x 10(-13) x and 6.9 ml/g, respectively. Other parameters calculated from these values indicate the shape of 5 S RNA is consistent with that of a prolate ellipsoid 160 A in length and 32 A wide. Sedimentation equilibrium results show that 5 S RNA exists as a monomer in the reconstitution buffer with an apparent molecular weight of 44,000. Ultraviolet absorption difference spectra show that approximately 75% of the bases in 5 S RNA are involved in base pairing, and of these base pairs 70% are G-C and 30% are A-U. These results on the overall shape and secondary structure of 5 S RNA have been incorporated with the results of other investigators as to the possible location of single-stranded and double-stranded helical regions, and a molecular model for 5 S RNA is proposed. The molecular model consists of three double helices in the shape of a prolate ellipsoid, with two of the double helical regions at one end of the molecule. The structure is consistent with the available data on the structure and function of 5 S RNA and bears similarity to the molecular model proposed by Osterberg et al. ((1976) Eur. J. Biochem. 68, 481-487) based on small angle x-ray scattering results and the secondary structure proposed by Madison ((1968) Annu. Rev. Biochem. 37, 131-148).