Non-covalent interactions in blue copper protein probed by Met16 mutation and electronic and resonance Raman spectroscopy of Achromobacter cycloclastes pseudoazurin

We have used low-temperature (77 K) resonance Raman (RR) spectroscopy as a probe of the electronic and molecular structure to investigate weak π-π interactions between the metal ion-coordinated His imidazoles and aromatic side chains in the second coordination sphere of blue copper proteins. For this purpose, the RR spectra of Met16 mutants of Achromobacter cycloclastes pseudoazurin (AcPAz) with aromatic (Met16Tyr, Met16Trp, and Met16Phe) and aliphatic (Met16Ala, Met16Val, Met16Leu, and Met16Ile) amino acid side chains have been obtained and analyzed over the 100-500 cm⁻¹ spectral region. Subtle strengthening of the Cu(II)-S(Cys) interaction on replacing Met16 with Tyr, Trp, and Phe is indicated by the upshifted (0.3-0.8 cm⁻¹) RR bands involving ν(Cu-S)_Cys stretching modes. In contrast, the RR spectra of Met16 mutants with aliphatic amino acids revealed larger (0.2-1.8 cm⁻¹) shifts of the ν(Cu-S)_Cys stretching modes to a lower frequency region, which indicate a weakening of the Cu(II)-S(Cys) bond. Comparisons of the predominantly ν(Cu-S)_Cys stretching RR peaks of the Met16X = Tyr, Trp, and Phe variants, with the molar absorptivity ratio ε₁/ε₂ of σ(∼455 nm)/π(∼595 nm) (Cys)S → Cu(II) charge-transfer bands in the optical spectrum and the axial/rhombic EPR signals, revealed a slightly more trigonal disposition of ligands about the copper(II) ion. In contrast, the RR spectra of Met16Z = Ala, Val, Leu, and Ile variants with aliphatic amino acid side chains show a more tetrahedral perturbation of the copper active site, as judged by the lower frequencies of the ν(Cu-S)_Cys stretching modes, much larger values of the ε₁/ε₂ ratio, and the increased rhombicity of the EPR spectra.     

Stabilization of protein structure through π–π interaction in the second coordination sphere of pseudoazurin

Noncovalent, weak interactions in the second coordination sphere of the copper active site of Pseudoazurin (PAz) from Achromobacter cycloclastes were examined using a series of Met16X variants. In this study, the differences in protein stability due to the changes in the nature of the 16th amino acid (Met, Phe, Val, Ile) were investigated by electrospray ionization mass spectrometry (ESI‐MS) and far‐UV circular dichroism (CD) as a result of acid denaturation. The percentage of native states (folded holo forms) of Met16Phe variants was estimated to be 75% at pH 2.9 although the wild‐type (WT), Met16Val and Met16Ile PAz, became completely unfolded. The high stability under acidic conditions is correlated with the result of the active site being stabilized by the aromatic substitution of the Met16 residue. The π–π interaction in the second coordination sphere makes a significant contribution to the stability of active site and the protein matrix.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Introduction of a pi-pi interaction at the active site of a cupredoxin: characterization of the Met16Phe Pseudoazurin mutant

The Met16Phe mutant of the type 1 copper protein pseudoazurin (PACu), in which a phenyl ring is introduced close to the imidazole moiety of the His81 ligand, has been characterized. NMR studies indicate that the introduced phenyl ring is parallel to the imidazole group of His81. The mutation has a subtle effect on the position of the two S(Cys)-->Cu(II) ligand-to-metal charge transfer bands in the visible spectrum of PACu(II) and a more significant influence on their intensities resulting in a A(459)/A(598) ratio of 0.31 for Met16Phe as compared to a A(453)/A(594) ratio of 0.43 for wild-type PACu(II) at pH 8. The electron paramagnetic resonance spectrum of the Met16Phe variant is more axial than that of the wild-type protein, and the resonance Raman spectrum of the mutant exhibits subtle differences. A C(gamma)H proton of Met86 exhibits a much smaller hyperfine shift in the paramagnetic (1)H NMR spectrum of Met16Phe PACu(II) as compared to its position in the wild-type protein, which indicates a weaker axial Cu-S(Met86) interaction in the mutant. The Met16Phe mutation results in an approximately 60 mV increase in the reduction potential of PACu. The pK(a) value of the ligand His81 decreases from 4.9 in wild-type PACu(I) to 4.5 in Met16Phe PACu(I) indicating that the pi-pi contact with Phe16 stabilizes the Cu-N(His81) interaction. The Met16Phe variant of PACu has a self-exchange rate constant at pH 7.6 (25 degrees C) of 9.8 x 10(3) M(-)(1) s(-)(1) as compared to the considerably smaller value of 3.7 x 10(3) M(-)(1) s(-)(1) for the wild-type protein under identical conditions. The enhanced electron transfer reactivity of Met16Phe PACu is a consequence of a lower reorganization energy due to additional active site rigidity caused by the pi-pi interaction between His81 and the introduced phenyl ring.     

Select nutrients in the ovine uterine lumen. I. Amino acids, glucose, and ions in uterine lumenal flushings of cyclic and pregnant ewes

Nutrients in uterine secretions are essential for development and survival of conceptuses (embryo and associated extraembryonic membranes) during pregnancy; however, little is known about changes in the amounts of specific nutrients in the uterine fluids of cyclic and pregnant ruminants. This study determined quantities of glucose, amino acids, glutathione, calcium, sodium, and potassium in uterine lumenal fluid from cyclic (Days 3-16) and pregnant (Days 10-16) ewes. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium, and sodium were greater in the uterine fluid of pregnant compared with cyclic ewes between Days 10 and 16. In cyclic ewes, only modest changes in the total amounts of glucose, Asn, Cit, Tyr, Trp, Met, Val, Cys, glutathione, calcium, and potassium were detected between Days 3 and 16. However, in pregnant ewes, amounts of glucose, Arg, Gln, Glu, Gly, Cys, Leu, Pro, glutathione, calcium, and potassium in uterine fluids increased 3- to 23-fold between Days 10 and 14 and remained high to Day 16. Of particular interest were increases in glucose, Arg, Leu, and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Total amounts of His, ornithine, Lys, Ser, Thr, Ile, Phe, Trp, Met, and Cit in uterine fluids also increased, but to a lesser extent during early pregnancy. These novel results indicate activation of pregnancy-associated mechanisms for transport of nutrients into the uterine lumen, and they provide a framework for future studies of nutrients, including glucose, amino acids, and glutathione, required to activate nutrient-sensing cell signaling pathways for growth, development, and survival of conceptuses, as well as for optimization of culture media for in vitro studies of conceptus development.     

New insight into the binding mode of peptides at urotensin‐II receptor by Trp‐constrained analogues of P5U and urantide

Urotensin II (U‐II) is a disulfide bridged peptide hormone identified as the ligand of a G‐protein‐coupled receptor. Human U‐II (H‐Glu‐Thr‐Pro‐Asp‐c[Cys‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U‐II termed P5U (H‐Asp‐c[Pen‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) and the compound termed urantide (H‐Asp‐c[Pen‐Phe‐d ‐Trp‐Orn‐Tyr‐Cys]‐Val‐OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp⁷ residue was replaced by the highly constrained l ‐Tpi and d ‐Tpi residues. The replacement of the Trp⁷ by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation–activity relationships previously reported on UT receptor ligands. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of Met297 in the seventh transmembrane segment of the tachykinin NK2 receptor with neurokinin A

We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.     

Comparative micelle structure. IV. The similarity between caprine alphas-casein and bovine alphas-3- casein.

The major component of caprine (goat) alphas-casein has been isolated by DEAE-and CM-cellulose chromatography in buffers containing urea and 2-mercaptoethanol. The protein has a molecular weight of 25700 as determined by gel filtration on Sepharose 6B in guanidine hydrochloride. Its composition, Asp17, Thr14, Ser14, Glu45, Pro18, Gly4, Ala10, Cys2, Val12, Met4, Ile12, Leu12, Tyr11, Phe8, His5, Lys22, Arg6, Trp2 and 7 phosphate residues, is much closer to that of bovine alphas3-casein than to bovine alphas1-casein. The caprin alphas-casein is more easily precipitated with Ca2+ than bovine alphas3-casein at 37 degrees C, pH 6.8, which in turn is more easily precipitated than bovine alphas1-casein.     

Studies on D-tetrose metabolism. VI. Crystallization and some properties of D-erythrulose reducatase from beef liver.

D-Erythrulose reductase of beef liver was crystallized from ammonium sulfate solution at pH 8.17. The crystals are needle-shaped. The enzyme protein contains 851 amino acid residues per mole of the enzyme: Lys28, His11, Arg52, Asp79, Thr58, Ser56, Glu68, Pro20, Gly80, Ala107, Val112, Met24, Ile31, Leu88, Tyr7, Phe22, Trp4, and Cys4. The enzyme is inactivated by exposure to temperatures below 12degrees. The inactivation is accelerated by increasing the salt concentration and decreasing the enzyme concentration. The pH of the medium also has a pronounced effect, the maximum stability of the enzyme is obtained at pH 8.5. NADP+ protected the enzyme from cold inactivation at all stages of the process and also afforded protection against inactivation by heat and pH. The cold inactivation of the enzyme is accompanied by dissociation of the enzyme protein to subunits.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Effect of amino acids on X-ray-induced hydrogen peroxide and hydroxyl radical formation in water and 8-oxoguanine in DNA

Generation of hydrogen peroxide and hydroxyl radicals in L-amino acid solutions in phosphate buffer, pH 7.4, under X-ray irradiation was determined by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system and using the fluorescent probe coumarin-3-carboxylic acid, respectively. Amino acids are divided into three groups according to their effect on the hydrogen peroxide formation under irradiation: those decreasing yield of H2O2, having no effect, and increasing its yield. All studied amino acids at 1 mM concentration decrease the yield of hydroxyl radicals in solution under X-ray irradiation. However, the highest effect is observed in the order: Cys > His > Phe = Met = Trp > Tyr. At Cys, Tyr, and His concentrations close to physiological, the yield of hydroxyl radicals decreases significantly. Immunoenzyme analysis using monoclonal antibodies to 8-oxoguanine (8-oxo-7,8-dihydroguanine) was applied to study the effect of amino acids with the most pronounced antioxidant properties (Cys, Met, Tyr, Trp, Phe, His, Lys, Arg, Pro) on 8-oxoguanine formation in vitro under X-ray irradiation. It is shown that amino acids decrease the content of 8-oxoguanine in DNA. These amino acids within DNA-binding proteins may protect intracellular DNA against oxidative damage caused by formation of reactive oxygen species in conditions of moderate oxidative stress.     

Quantitative determination of aromatic amino acids and related compounds in rumen fluid by high-performance liquid chromatography

A rapid method for the quantitative determination of tyrosine (Tyr), phenylalanine (Phe), p-hydroxybenzoic acid (HBA), p-hydroxyphenylacetic acid (HPA), benzoic acid (BZA), p-hydroxyphenylpyruvic acid (HPY), phenylacetic acid (PAA), phenyllactic acid (PLA), tryptophan (Trp), indoleacetic acid (IAA), phenylpyruvic acid (PPY), phenylpropionic acid (PPA) and cinnamic acid (CNA) in goat rumen fluid was established by high-performance liquid chromatography (HPLC). The mobile phase used for isocratic elution was 50 mM sodium phosphate buffer (pH 6.5)–methanol (97:3, v/v). The flow-rate was 1.0 ml/min; column temperature 40°C and compounds were monitored at 215 nm with a UV absorbance detector after injection of 10 μl of filtered rumen fluid. Analysis was completed within 40 min. The minimum detectable limits of quantification (μM) of these compounds were Tyr, 2; Phe, 3; HBA, 1; HPA, 2; BZA, 2; HPY, 8; PAA, 3; PLA, 4; Trp, 2; IAA, 2; PPY, 15; PPA, 8 and CNA, 4. Detectable levels of Tyr, Phe, HPA, BZA, HPY, PAA, PLA, Trp and PPA were found in the deproteinized rumen fluid of goat fed a haycube and concentrate mixture. PAA was the predominant compound before and after feeding. The concentrations of HPA, BZA, PAA, PLA and PPA in the goat rumen fluid increased after feeding, while the concentration of Tyr decreased. Phe, HPY and Trp were minor components at all times. PPY, IAA and CNA were not detected and HBA was not completely resolved in the goat rumen fluid.     

Formation of mutagens by amino-carbonyl reactions

The formation of mutagens by amino-carbonyl reactions of 20 kinds of amino acid and sugars after heating at 100 degrees C for 10 h was examined by the Ames test. The browned solutions of Gly, Ala, Val, Leu, Ile, Ser, Thr, Gln, Lys X HCl, Arg, Phe, Cys, Met and Pro with Glc caused mutation of Salmonella typhimurium TA100 and/or TA98 with or without S9 mix. The presence of S9 mix increased the mutagenic activity of the browned solutions of Cys and Phe with Glc on TA100 and of those of Gly, Ala, Val, Ile and Cys on TA98, but decreased the activity of other solutions. No revertants of Salmonella were induced by the browned solutions of Trp, Tyr, Asp, Asn, Glu and (Cys)2 with Glc. Among positive browned solutions, Cys, Lys, Arg and Phe had the stronger activity, but their activity was weak compared with that of pyrolysates or chemical mutagens such as Trp-P-1, Trp-P-2 and 4-nitroquinoline-N-oxide. The mutagenic activity of the browned solutions increased with prolongation of heating time and varied with the pH of the reaction mixture. Fru, Gal, Ara, Xyl, Man, Lac and Suc also had the ability to form mutagens in the browning reactions with amino acids.     

SH-proteinase from bean Phaseolus vulgaris var. Perlicka.

An SH-proteinase (EC 3.4.22.-) has been isolated from beans of the species Phaseolus vulgaris var. Perlicka. The enzyme is homogeneous when subjected to disc electrophoresis, electrofocusing and sedimentation analysis. The molecular weight was determined as 26,000-28,000 by gel filtration, 30,850 +/- 1500 by sedimentation analysis and 26,930-27,410 by calculation from the amino acid composition (Lys20-21, His3, Arg9, Asp21-22, Thr13, Ser18, Pro12-13, Glu23-24, Gly30, Ala16, Cys/29, Val19, Met1, Ile10, Leu13, Tyr14, Phe6, Trp3). The N-terminal amino acid of the proteinase is isoleucine. The effect of concentration, time of hydrolysis, pH, temperature, cations, anions, urea and guanidine - HCl on the proteolytic activity of the SH-proteinase was studied.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

Interactions between hydrophobic side chains within alpha-helices.

The thermodynamic basis of helix stability in peptides and proteins is a topic of considerable interest. Accordingly, we have computed the interactions between side chains of all hydrophobic residue pairs and selected triples in a model helix, using Boltzmann-weighted exhaustive modeling. Specifically, all possible pairs from the set Ala, Cys, His, Ile, Leu, Met, Phe, Trp, Tyr, and Val were modeled at spacings of (i, i + 2), (i, i + 3), and (i, i + 4) in the central turn of a model poly-alanyl alpha-helix. Significant interactions--both stabilizing and destabilizing-- were found to occur at spacings of (i, i + 3) and (i, i + 4), particularly in side chains with rings (i.e., Phe, Tyr, Trp, and His). In addition, modeling of leucine triples in a helix showed that the free energy can exceed the sum of pairwise interactions in certain cases. Our calculated interaction values both rationalize recent experimental data and provide previously unavailable estimates of the constituent energies and entropies of interaction.     

Lysozyme modification by the fenton reaction and gamma radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H2 O2) or hydroxyl radicals produced by y radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. Gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.     

Detailed characterization of an anti-factor IX monoclonal antibody that neutralizes the prolonged ox brain prothrombin time of hemophilia B(M) by synthetic peptides

In a previous study, we prepared a monoclonal antibody (MoAb) to coagulation factor IX (FIX), designated 65-10, which interfered with the activation of FIX by the activated factor XI/Ca(2+) and neutralized the prolonged ox brain prothrombin time of hemophilia B(M) [11,12]. The location of the epitope on the FIX for 65-10 MoAb is (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) [21]. In this paper, we studied in more detail an epitope on FIX using the systematic substitution of different amino acids at each residue of the epitope peptides and the influence of the epitope peptide on the prolonged ox brain prothrombin time of the hemophilia B(M) plasma of 65-10 MoAb. In the replacement set of amino acids, peptides showing low or no reactivity to 65-10 were (175)Phe --> Asp, Glu, Gly, Lys, Arg, Thr, Val, (176)Asn --> Asp, Glu, Phe, Ile, Lys, Leu, Pro, Val, Tyr, (177)Asp --> Cys, Glu, Phe, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, and (178) Phe --> Pro. These results imply that a hydrophobic molecule of (175) Phe, a hydrophilic molecule of (176)Asn, and a negative charge molecule of (177)Asp were important to the epitope. The 65-10 MoAb antibody neutralized the prolonged ox brain prothrombin time of hemophilia B(M) Nagoya 2 ((180)Arg -->Trp) and Kashihara ((181)Val --> Phe) as well as B(M) Kiryu ((313)Val --> Asp) and Niigata ((390)Ala --> Val). This reaction was inhibited by preincubation with a (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) peptide conjugated with bovine serum albumin (BSA). 65-10 MoAb that has been useful in detailing epitopes will be useful for qualitative analysis of hemophilia B(M).     

The intrinsic fluorescence of the recombinant human leukocyte interferon-alpha A and fibroblast interferon-beta 1

The environment of Trp residues of the recombinant human interferons has been studied by the analysis of the emission spectra of native and denatured proteins at pH 1.5-8.5 and temperature 10-65 degrees C in the presence and absence of the anionic, cationic and neutral to charge contact quenchers--KI, CsCl and acrylamide, respectively. The obtained data allow to suppose that in IFN-alpha A and IFN-beta 1 Trp141 interacts with Arg145 and one or several from the following residues--Phe124, Ile127, Tyr130, Leu131, whereas Trp77--with Arg33 and Phe36, Phe78, Leu81 or Leu82 (Ile81 or Val82 for IFN-beta 1).     

Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.