Difference Spectral Studies Research Articles

Clavulanic Acid Inactivation of SHV-1 and the Inhibitor-resistant S130G SHV-1 β-Lactamase: INSIGHTS INTO THE MECHANISM OF INHIBITION

Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1beta-lactamases, enzymes that confer resistance to beta-lactams in many gram-negative pathogens. This compound has enjoyed widespread clinical use as part of beta-lactam beta-lactamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 beta-lactamase significantly compromise the efficacy of this combination. A single amino acid change at Ambler position Ser130 (Ser --> Gly) results in resistance to inactivation by clavulanate in the SHV-1 and TEM-1beta-lactamases. Herein, we investigated the inactivation of SHV-1 and the inhibitor-resistant S130G variant beta-lactamases by clavulanate. Using liquid chromatography electrospray ionization mass spectrometry, we detected multiple modified proteins when SHV-1 beta-lactamase is inactivated by clavulanate. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to study tryptic digests of SHV-1 and S130Gbeta-lactamases (+/- inactivation with clavulanate) and identified peptides modified at the active site Ser70. Ultraviolet (UV) difference spectral studies comparing SHV-1 and S130Gbeta-lactamases inactivated by clavulanate showed that the formation of reaction intermediates with absorption maxima at 227 and 280 nm are diminished and delayed when S130Gbeta-lactamase is inactivated. We conclude that the clavulanic acid inhibition of the S130G beta-lactamase must follow a branch of the normal inactivation pathway. These findings highlight the importance of understanding the intermediates formed in the inactivation process of inhibitor-resistant beta-lactamases and suggest how strategic chemical design can lead to novel ways to inhibit beta-lactamases.

Peptide Ribonucleic Acids (PRNA). 2. A Novel Strategy for Active Control of DNA Recognition through Borate Ester Formation

The effect of adding borax and boric acids on the nucleobase orientation and recognition behavior of novel mono- and oligomeric peptide ribonucleic acids (PRNAs) has been investigated. The base orientation of 5‘-amino-5‘-deoxyuridine and 5‘-amino-5‘-deoxycytidine was shown by CD and NOE difference spectral studies to switch from anti to syn in borate buffer or upon addition of borax. The origin of this phenomenon is elucidated to be the cooperative effect of the cyclic borate esterification of the sugar's cis-2‘,3‘-diol and the hydrogen-bonding interaction between the sugar's 5‘-amino proton and the base's 2-carbonyl oxygen. Because this new strategy for switching the base orientation through the addition of borate is potentially applicable to the recognition control of nucleic acids if the sugar's 5‘-proton and cis-2‘,3‘-diol remain unmodified, we synthesized a series of PRNAs, in which the 5‘-amino-5‘-deoxypyrimidine ribonucleoside moiety was appended to a mono- or oligo(γ-l-glutamic acid) backbone thro...

Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS-PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an Mr of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI-trypsin/chymotrypsin complexes by difference spectral studies. Apparent Ka values of complexes of inhibitor with trypsin and chymotrypsin were 2.1x10(7) M(-1) and 3.1x10(7) M(-1), respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.

Ultraviolet Difference Spectral Studies in the γ-radiolysis of DNA and Model Compounds. II. Oxygen and Temperature Dependence in Adenine Radiation Chemistry

Ultraviolet difference spectra between gamma-irradiated and unirradiated aqueous solutions of oxygenated adenine reveal spectral maxima at 224, 286 and 360 nm, and suggest the existence of multiple radiation products. The presence of long-wavelength absorbing products implies the extension of the conjugation of the adenine ring. The product absorbing at 360 nm appears only in the presence of oxygen, with its yield dependent on both oxygen concentration and temperature. The absorbance peak at 360 nm in the UV difference spectra appears to demonstrate simple first-order decay kinetics, with a half-life of 50 min at 3 degrees C. At temperatures below 23 degrees C relative yields of the radiation products are approximately constant, suggesting a common mechanism. Higher temperatures apparently induce a mechanistic alteration. Since the temperature during and subsequent to irradiation, as well as the oxygen concentration of the solution, affects the absolute yields and distribution of the radiation products, great care must be exercised in controlling these variables.

Ultraviolet Difference Spectral Studies in the Gamma Radiolysis of DNA and Model Compounds. I. Aqueous Solutions of DNA Bases

Ultraviolet difference spectra of gamma-irradiated, air-saturated aqueous solutions of DNA bases vs. unirradiated solutions of the same bases are shown to be a very sensitive supplemental tool with which to investigate the yields, postirradiation kinetics, and general nature of DNA base radiation products. Irradiated pyrimidines yield difference spectra which are approximately negative mirror-images of the base absorption spectra in the near-UV, indicating loss of ring conjugation. Difference spectra of irradiated purines yield a more complex pattern containing a positive long-wavelength peak, interpreted as radiation-induced extension of conjugation of the pi electron system beyond that of the unirradiated purine. On the basis of the spectroscopic evidence from these studies, 8-hydroxyguanine appears to be the dominant UV-absorbing radiation product in air-saturated guanine solutions with a G-value of 0.3 molec (100 eV)-1. Difference spectral studies provide isosbestic points which can be used in testing proposed radiation products and their yields. Such spectral studies are a rapid, non-invasive, supplemental tool which can be employed in conjunction with other analytical techniques in radiation-chemical studies, and which is one of the few tools able to detect short-lived chemical intermediates observed in oxygenated solutions of irradiated purines.

Interaction of allylisothiocyanate with bovine serum albumin

The interaction of allylisothiocyanate with bovine serum albumin was monitored by “available” lysine estimation, electrometric and spectrophotometric titration, u.v. absorption and difference spectroscopy and electrophoresis. “Available” lysine estimation and electrometric titration indicated that the interaction increased with the reaction pH. Spectrophotometric titration indicated that at least two of the tyrosine groups became masked, probably via H‐bonding, upon interaction of the protein with allylisothiocyanate. The pKint values of the unreacted ∍‐amino groups of lysine and the titrable phenolic groups of tyrosine did not alter upon interaction. The electrophoretic mobility of the protein increased with the interaction. Difference spectral studies suggested thiourea formation.

Binding site amino acid residues of jack fruit (artocarpus integrifolia) seed lectin: chemical modification and protein difference spectral studies

The effect of chemical modification of amino acid residues essential for sugar binding in the α-D-galactoside specific jack fruit (Artocarpus integrifolia) seed lectin and the protection of the residues by specific sugar from modification were studied. Citraconylation or maleylation of 75 % of its lysyl residues or acetylation of 70 % of the tyrosyl residues completely abolished sugar binding and agglutination without dissociation of subunits. 1-O- methyl α-D-galactoside could protect its essential lysyl and tyrosyl groups from modification. Tryptophan could not be detected in the protein. Difference absorption spectra on binding of the above sugar confirmed the role of tyrosine residues and showed an association constant K = 0·4 × 10 3 M -1 . Data suggests that the lectin could be immobilized without any loss of sugar binding activity.

Ultraviolet difference spectral studies of human serotransferrin and lactotransferrin

In order to determine the conformational relationship of iron binding of human serotransferrin and lactotransferrin, ultraviolet difference spectral studies were performed in the presence of guanidine chloride and perturbants as deuterium oxide, ethylene glycol, glycerol and polyethylene glycol. In the presence of guanidine chloride solution the molar absorption differences at 292 nm of iron-saturated forms versus iron-free forms of human serotransferrin and lactotransferrin are respectively −16000 ± 1000 and −14000 ± 775 . These modifications may be attributed to the involvement of tryptophan residues in the iron-binding sites of the two proteins. However, the results do not demonstrate that these tryptophan residues are bound directly to iron. Difference spectral studies in the presence of perturbants show that the apparent exposed tryptophan and tyrosine residues are higher with shorter range perturbants in iron-free forms of both transferrin molecules. The most important modification of exposed tyrosine residues has been noticed upon removing iron from human lactotransferrin than from serotransferrin.

On the structure of the cofactor in the complex formed with thymidylate synthetase, 5,10-methylenetetrahydrofolate and 5-fluoro-2′-deoxyuridylate

A study was undertaken to ascertain whether dihydrofolic acid is produced in the complex formed with 5,10-methylenetetrahydrofolate, 5-fluorodeoxyuridylate and thymidylate synthetase, as suggested by ultraviolet difference spectral studies. The complex was formed using the cofactor specifically labeled with tritium at the 6-position. After dissociation by equilibration with unlabeled cofactor, it was demonstrated that the tritium remained exclusively at the 6-position. Had oxidation of the cofactor occurred within the complex to give a methylated enzyme form, tritium should have been transferred to the one-carbon unit of the cofactor. It was also found that the difference spectrum of the ternary complex which resembles that of dihydrofolic acid can also be produced by substituting an analog of the cofactor which is not susceptible to oxidation. The results described here demonstrate that oxidation of the cofactor does not occur in the ternary complex and suggest that the unusual ultraviolet spectrum results from perturbations of a chromophore of the bound cofactor.

Nuclear magnetic resonance and ultraviolet difference spectral studies of the binding properties of turkey egg white lysozyme: Consequences of the replacement of ASP 101 by glycine

The nuclear magnetic resonance spectrum of the 19F nuclei in N-trifluoroacetylated chitotriose was studied in the presence of turkey lysozyme. In contrast to results previously obtained with hen lysozyme, the 19F nmr spectrum of the complex did not show any striking pH dependence. It was, in fact, very similar at all pH's to the spectrum of the trisaccharide complexed with hen lysozyme at low pH, where Asp 101 is protonated. The replacement of Asp 101 in turkey lysozyme by a glycine is thought to account for this difference and the results allow unequivocal assignment of a value of 4.2 to the pK a of Asp 101 in hen lysozyme. The dissociation constant of the chitotriose-turkey lysozyme complex was measured at various pH's using uv difference methods and compared with that previously reported for the hen lysozyme-chitotriose complex. Again, the results could be attributed to the loss in binding energy due to the absence of Asp 101. In contrast to chitotriose, the binding of chitobiose and methyl-2-acetamido-2-deoxy-β- d-glucopyranoside as studied by both uv difference and nmr methods is the same within experimental error for turkey and hen lysozyme. The results obtained for binding of chitobiose suggest that Asp 101 does not contribute as much to the binding energy of the disaccharide as was previously thought. Finally, the specific activities of both of these lysozymes against Micrococcus lysodeikticus were found to be identical.

Difference spectral studies on the interactions between ribonuclease T and its substrate analogues.

Journal Article Difference Spectral Studies on the Interactions between Ribonuclease T1 and Its Substrate Analogues Get access Tairo OSHIMA, Tairo OSHIMA The Department of Agricultural Chemistry, Faculty of Agriculture, The University of TokyoBunkyo-ku, Tokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar Kazutomo IMAHORI Kazutomo IMAHORI The Department of Agricultural Chemistry, Faculty of Agriculture, The University of TokyoBunkyo-ku, Tokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 69, Issue 5, May 1971, Pages 987–990, https://doi.org/10.1093/oxfordjournals.jbchem.a129551 Published: 01 May 1971 Article history Received: 18 March 1971 Published: 01 May 1971

Circular dichroism studies on concanavalin A

Optical rotatory dispersion and circular dichroism studies on native and demetallized concanavalin A indicate that this protein consists essentially of a mixture of β-form and random coil. The induction of α-helix in the native protein is accomplished through the use of 2-chloroethanol. The sequential addition of transition metal, Ca 2+ and α-methyl-D-glucopyranoside to solutions of demetallized protein results in small but reproducible alterations in the aromatic CD spectrum, the largest changes being noted with calcium addition. Aromatic CD difference spectral studies suggest that a single transition may be involved in the binding of metal and α-MG to the protein; the wavelength position of this transition implicates tyrosine residues. At the same time, no significant variations occur in the secondary structure of the protein as reflected by the constancy of the far ultraviolet CD spectrum with ligand complexation. Exploration of the CD aromatic fine structure as a function of pH indicates that both tyrosine and tryptophan chromophores are involved.

Conformational studies on the tryptic digestion fragments of human immunoglobulin G

Conformational studies on Fab(t) and Fc(t) (normal and myeloma) have been carried out by circular dichroism, difference spectra, sedimentation, and viscosity measurements. Both Fab(t) and Fc(t) were largely unfolded in acid (pH 2.1) and alkali (pH 11.7), and the unfolding was greater in alkali than in acid for Fab(t). However, for Fc(t) the circular dichroism spectral change was slightly higher in acid (pH 2.1) than in alkali (pH 11.7). It was also noted that the disorganizations were not complete under these conditions. Similar types of disorganizations were observed in heat denatured Fab(t) and Fc(t). Difference spectral studies demonstrated the presence of a significant number of buried tyrosine and tryptophan residues in both fragments, and a large proportion of the residues became exposed on acid denaturation. The results led us to conclude that both Fab(t) and Fc(t) may be characterized by structures of different stabilities. There seem to be small stable structured regions containing some of the β structures while the other portions are more susceptible to denaturation. Also, there are regions in these proteins which are rich in hydrophobic interactions. Sodium dodecyl sulfate refolded both Fab(t) and Fc(t) into partial helical structures, although the accessibility and rotational freedom of the side chain aromatic chromophores were increased. In 6 m GuHCl containing 0.1 m 2-mercaptoethanol, complete disorganization of Fab(t) was demonstrated despite the fact that its circular dichroism spectra around 217 nm differed from the spectra of random coil polyamino acids. In spite of certain differences, the behavior of Fab(t) and Fc(t) towards different denaturing agents was similar pointing out homology in their basic structural features.

Ultraviolet difference spectrl studies of conalbumin complexes with transition metal ions.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTUltraviolet difference spectral studies of conalbumin complexes with transition metal ionsA. T. Tan and Robert C. WoodworthCite this: Biochemistry 1969, 8, 9, 3711–3716Publication Date (Print):September 1, 1969Publication History Published online1 May 2002Published inissue 1 September 1969https://doi.org/10.1021/bi00837a033RIGHTS & PERMISSIONSArticle Views231Altmetric-Citations116LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (620 KB) Get e-Alerts Get e-Alerts