A comparative molecular field analysis of the biotransformation of sulfides by Rhodococcus erythropolis

A comparative molecular field analysis (CoMFA) was used to model the efficacy with which the Rhodococcus erythropolis mono-oxygenase, DszC, catalyzes the enantioselective sulfoxidation of a broad range of substrates. Experimentally determined values of both the yield and enantiomeric excess for this reaction were employed to create these CoMFA models. A highly predictive CoMFA model was constructed for the prediction of enantiomeric excess of the sulfoxide product. The predictive ability of the model was demonstrated by both cross-validation of the training set (q² = 0.74) and for an external test set of substrates. The enantiomeric excesses of the members of the test set, which also included two amino acid sulfides that were structurally distinct from the membership of the training set, were predicted well by the CoMFA model. Product yield was not modelled well by any CoMFA model. Different models comparing the likely bioactive conformations of the substrates suggest that most compounds assume an ‘extended’ conformation upon binding. Contour diagrams illustrating significant substrate–enzyme interactions suggest that the model, which predicts the enantiomeric excess, is consistent with previous conclusions regarding the effect of various substrate substitutions on the enantiopurity of the product of the biotransformation.     

Enantiomeric separation of ornithine in complex mixtures of amino acids by EKC with off-line derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

A new analytical methodology was developed by EKC enabling the fast enantiomeric separation of Ornithine in complex mixtures of amino acids. A previous derivatization step with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was achieved to enable the sensitive UV detection of amino acids as well as to make possible their interaction with the CDs employed as chiral selectors. A dual CD system containing an anionic and a neutral CD in phosphate buffer at acid pH showed a high resolving power allowing the enantiomeric separation of 18 protein amino acids and Orn. The method was applied to the analysis of fermented foods to investigate the extent of the presence of Orn enantiomers.     

Evaluation of the concentration and enantiomeric purity of selected free amino acids in fermented malt beverages (beers)

Even though amino acids are important trace components in the brewing of beers, they have not been extensively evaluated in these beverages. Studies involving the enantiomeric composition of these amino acids are even less prevalent. A brief summary of the brewing process for malt beverages is given. The total concentration and enantiomeric composition of three amino acids (leucine, phenylalanine, and proline) were determined in 25 different beers. Proline tended to have the highest average absolute concentration and the lowest percentage of the D -enantiomer in most samples. In some cases the relative amounts of D -phenylalanine and D -leucine exceeded 10% of the individual amino acids. The enantiomeric composition of the amino acids in different beer samples did not vary as extensively as the absolute concentrations. The reason for the concentration differences between proline and the other amino acids is discussed. A knowledge of amino acid concentrations and enantiomeric compositions appears to be useful in characterizing specific beers and brewing processes. © 1996 Wiley-Liss, Inc.     

Antibodies directed against L and D isovaline using a chemical derivatizing reagent for the measurement of their enantiomeric ratio in extraterrestrial samples: first step-production and characterization of antibodies

Determining the enantiomeric ratio of amino acids in meteorites requires very sensitive and precise measurements. In this study, an immunochemical approach, combined with new chemical derivatizing agents, was investigated for the measurement of the enantiomeric ratio of isovaline. In the initial step, L and D isovaline were derivatized with epsilon-benzyloxycarbonyl-L-lysine-(t-butyl ester)-chloroethylnitrosourea (Z-L-Lys-(OtBu)-CENU). The Z group was hydrolyzed and the resulting isovaline derivatives (L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) were conjugated with protein using glutaraldehyde and reduced with sodium borohydride. Rabbits were immunized with the immunogenic conjugates thus obtained. Antibodies were characterized using many compounds, both derivatized and underivatized, in competitive ELISA tests. These competition experiments performed enabled us to establish the following results: 1) unconjugated L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline were poorly recognized; 2) all related L-Lys(OtBu)-alpha-hydrogenated amino acids (L and D) were not recognized at all, which eliminates the possibility of the measurements being distorted by contamination; 3) only conjugated L-Lys(OtBu)-alpha-amino-isobutyric acid (AIB) was recognized by the antibody, 4) the enantiomeric discrimination of L and D isovaline through their derivatives (diastereoisomeric L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) was in accordance with the measurement of their enantiomeric ratio. Immunopurification was shown to enhance antibody specificity. The strategy employed shows potential for the quantification of meteoritic amino acids.     

Characterization of protein aggregation: the case of a therapeutic immunoglobulin

In this paper, a therapeutic immunoglobulin (Antibody A) has been characterized in two solutions: (1) 0.1% acetic acid containing 50 mM magnesium chloride, a solution in which the immunoglobulin is stable, and (2) 10 mM sodium phosphate buffer pH approximately 7. The protein solutions were characterized by microscopy, asymmetrical flow field-flow fractionation (FFF), light scattering, circular dichroism, fluorescence and fluorescence lifetime spectroscopy. The results show that Antibody A dissolved in 0.1% acetic acid containing 50 mM magnesium chloride exists as 88% monomer, 2% low molecular weight aggregates and 10% high molecular weight aggregates (>1 million Dalton). In phosphate buffer, Antibody A formed micrometre-sized aggregates that were best characterized by fluorescence microscopy. The aggregation of Antibody A in phosphate buffer was shown to be concomitant with conformational changes in amino acid residue side chains. The aggregates formed in phosphate buffer were easily disrupted during FFF analysis, indicating that they are formed by weak interactions. The combination of microscopy, asymmetrical flow field-flow fractionation (FFF) and spectroscopy allowed a reliable assessment of protein self association and aggregation.     

Molecular recognition in synthetic polymers. Enantiomeric resolution of amide derivatives of amino acids on molecularly imprinted polymers

Molecular imprints were prepared using L-phenylalanine anilide as the print molecule and methacrylic acid as the functional monomer. Methacrylic acid interacts ionically with the primary amine of the print molecule and via hydrogen bonding with the amide function. In the HPLC mode such polymers were shown to exhibit efficient enantiomeric resolution of a racemic mixture of the original print molecule. Enantiomeric resolution was shown to be dependent on the ratio of methacrylic acid to print molecule in the pre-polymerization mixture and specific for the presence of both print molecule and functional monomer. Further analyses showed the importance of both the primary amino and amide functions in the correct stereochemistry for recognition and enantiomeric resolution of compounds on such polymers. Other amide derivatives of amino acids including p-nitroanilides, beta-naphthylamides and amides were recognized by such polymers, and enantiomeric resolution was obtained for amide derivatives of amino acid ranging from alanine to tryptophan on a single polymer. The implications of these findings with respect to the mechanism of recognition and the ability to predict enantiomeric resolution of molecules on molecularly imprinted polymers will be discussed.     

ATP-dependent dissociation of non-disulfide-linked aggregates of coagulation factor VIII is a rate-limiting step for secretion

Deficiency in coagulation factor VIII leads to the bleeding disorder hemophilia A. Previous studies demonstrated that factor VIII secretion is limited due to an ATP-requiring step early in the secretory pathway. In this report, we identified that this ATP-dependent rate-limiting step involves the dissociation of non-disulfide-linked aggregates within the endoplasmic reticulum (ER). In contrast to the numerous examples of interchain disulfide-linked aggregates, factor VIII is the first protein characterized to form non-disulfide-linked high molecular weight aggregates within the ER. Approximately a third of newly synthesized factor VIII was detected in high molecular weight aggregates. These aggregates disappeared over time as functional factor VIII appeared in the medium. The aggregated complexes did not require proteasomal degradation for clearance. Aggregate formation was enhanced by ATP depletion, and upon restoration of metabolic energy, these aggregates were dissociated and secreted. With the coexpression of von Willebrand factor (vWF), a small portion of vWF coaggregated with factor VIII. However, vWF dissociated from the aggregates more rapidly than factor VIII, supporting that these aggregates are dynamic. An increase in the factor VIII expression level elicited a corresponding increase in the fraction of factor VIII that was aggregated. In addition, a 110 amino acid sequence containing a hydrophobic beta-sheet within factor VIII was identified that may predispose factor VIII to aggregation. These data show that formation and ATP-dependent dissolution of nondisulfide-linked factor VIII aggregates is a dynamic, rate-limiting step during the folding process in the early secretory pathway. In summary, we have identified an unprecedented requirement for protein transport out of the ER that involves an ATP-dependent dissociation of non-disulfide-linked aggregates within the ER.     

Determination of enantiomeric composition by negative-ion electrospray ionization-mass spectrometry using deprotonated N-(3,5-dinitrobenzoyl)amino acids as chiral selectors

The ability to use mixtures of deprotonated N-(3,5-dinitrobenzoyl)amino acids as chiral selectors for the determination of enantiomeric composition by electrospray ionization-mass spectrometry is demonstrated. For each experiment, two N-(3,5-dinitrobenzoyl)amino acids were chosen such that each would have opposite selectivity for the enantiomers of the analyte. Electrospray ionization-mass spectrometry, monitored in the negative ion mode, of solutions containing the two N-(3,5-dinitrobenzoyl)amino acids, sodium hydroxide, and the analyte, in a one-to-one mixture of methanol and water, afford peaks in the mass spectrum that correspond to the deprotonated 1:1 analyte-selector complexes. The ratio of the intensities of the complexes in the mass spectrum can be related to the enantiomeric composition of the analyte. Additionally, the sense and extent of chiral recognition is consistent with chromatographic observations, using chiral stationary phases derived from N-(3,5-dinitrobenzoyl)amino acids. Each analysis of enantiomeric composition requires less than 10 s to complete, indicating that this method has great potential for the development of fast-/high-throughput chiral analyses.     

Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols

The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).     

Determination of molecular species of enantiomeric diacylglycerols by chiral phase high performance liquid chromatography and polar capillary gas-liquid chromatography

A simple method is described for the determination of molecular species of enantiomeric sn-1,2- and sn-2,3-diacylglycerols derived from natural triacylglycerols by Grignard degradation. The method is based on a preparative separation of the enantiomeric diacylglycerols as 3,5-dinitrophenylurethane (DNPU) derivatives by high performance liquid chromatography (HPLC) on a chiral column (25 cm x 4.6 mm ID) containing R-(+)-1-(1-naphthyl)ethylamine as a stationary phase. This is followed by polar capillary gas-liquid chromatography (GLC) of the trimethylsilyl (TMS) ether derivatives of the enantiomeric diacylglycerols derived from the DNPU derivatives using trichlorosilane, which does not cause acyl migration and racemization during the reaction. The cleavage is better than 94% complete. The method was standardized with synthetic sn-1,2- and sn-2,3-dipalmitoyl- and rac-1,2-dioleoylglycerols and was applied to the identification and quantitation of individual molecular species of enantiomeric diacylglycerols generated by Grignard degradation of the triacylglycerols from corn oil, cocoa butter, and lard.     

Universal dissymmetry and the origin of biomolecular chirality

Handed systems are distributed over four general domains. These span the fundamental particles, the molecular enantiomers, the crystal enantiomorphs, and the spiral galaxies. The characterisation of the molecular enantiomers followed from the identification of the crystal enantiomorphs and revealed a chiral homogeneity in the biomolecules of the organic world. The origin of the homogeneity has been variously ascribed to a universal dissymmetric force, from Pasteur, or to a chance choice of the initial enantiomer perpetuated by the stereoselection of diastereomer production with recycling, from Fischer's "key and lock" hypothesis. The classical chiral fields identified by Curie require a particular time or location on the Earth's surface for a determinate molecular enantioselection, as do the weak charged current agencies of the non-classical weak interaction. The weak neutral current of the electroweak interaction provides a constant and uniform chiral agency which favours both the L-series of amino acids and polypeptides and the parent aldotriose of the D-series of sugars. The enantiomeric bias of the electroweak interaction is small at the molecular level: it may become significant either as a trigger-perturbation guiding the transition from a metastable autocatalytic racemic process to one of the two constituent enantiomeric reaction channels, or by cumulative amplification in a large chirally-homogeneous aggregate of enantiomer units.     

The radiolysis and radioracemization of poly-L-leucines.

The brief history of the discovery of radioracemization, the racemization of an optically active substance induced by ionizing radiation, is reviewed. Our early studies involving the radiolysis and radioracemization of D- and L-leucine using gamma radiation from a 111-TBq 60Co gamma-ray source are described briefly, as are later experiments involving other protein amino acids and their salts, as well as the nonprotein amino acid, isovaline. The implications of the results of such studies for the Vester-Ulbricht mechanism which proposes longitudinally polarized beta radiation as the origin of molecular chirality, for the cosmological question of the enantiomeric compositions of amino acids in the Murchison meteorite, and for the use of D/L ratios of amino acids for geochronological and geothermal estimates are reviewed briefly. These past radiolysis-radio- racemization studies have involved only monomeric amino acids. The present research, extending such investigations to two homochiral L-leucine polypeptides, (L-Leu)10 and (L-Leu)78, was undertaken to see if a polymer of an amino acid might be more stable to radiolysis and radioracemization than the corresponding monomer. It was found that these polypeptides were more stable to radiolysis than was the leucine monomer, but that the extents of radioracemization in all samples were comparable.     

Studies on the apoproteins of the major lipoprotein of the yolk of hen's eggs. I. Isolation and properties of the low-molecular-weight apoproteins.

In a continuing study of protein-lipid interactions in egg yolk, the total apoprotein mixture (i.e. the 'apovitellenins') from the high-lipid, low-density lipoprotein (density 0.97 g/ml) of the yolk from hen's eggs has been isolated in a soluble form. By gel-filtration chromatography in 6M urea the mixture has been separated into several fractions from which three new low-molecular-weight proteins (I, Ia, and II), making up about 30% of the total, have been isolated. The most plentiful of these (I) consists of stable aggregates with several identical subunits each of molecular weight about 10 000. This protein is analogous to the principal protein from the corresponding lipoprotein of emu's egg yolk, i.e. emu's apovitellenin I. Hen's apovitellenin I has a slightly different amino acid composition from that of the emu; notably it contains a sulphydryl group. The hen's protein also forms more stable aggregates that are dissociated by detergent and by guanidine hydrochloride but are stable in urea. The molecular weight of Ia is similar to that of I and the amino acid composition is the same, with the exception that Ia has a higher proportion of amide groups. It aggregates less readily than I under the same conditions. The third new protein (II, 'hens's apovitellenin II') has a molecular weight of about 20 000. It has no tyrosine or methionine residues, but contains glucosamine and has several disulphide groups. It has been isolated in very small amount only.     

Catalytic effects of glycine on prebiotic divaline and diproline formation

The catalytic effects of the simple amino acid glycine on the formation of diproline and divaline in the prebiotically relevant salt-induced peptide formation (SIPF) reaction was investigated in systems of different amino acid starting concentrations and using the two enantiomeric forms of the respective amino acid. Results show an improved applicability of the SIPF reaction to prebiotic conditions, especially at low amino acid concentrations, as presumably present in a primordial scenario, and indicate excellent conditions and resources for chemical evolution of peptides and proteins on the early earth. For valine, furthermore differences in catalytic yield increase are found indicating a chiral selectivity of the active copper complex of the reaction and showing a connection to previously found enantiomeric differences in complex formation constants with amino acids.     

Fast mass spectrometry-based enantiomeric excess determination of proteinogenic amino acids

A rapid determination of the enantiomeric excess of proteinogenic amino acids is of great importance in various fields of chemical and biologic research and industries. Owing to their different biologic effects, enantiomers are interesting research subjects in drug development for the design of new and more efficient pharmaceuticals. Usually, the enantiomeric composition of amino acids is determined by conventional analytical methods such as liquid or gas chromatography or capillary electrophoresis. These analytical techniques do not fulfill the requirements of high-throughput screening due to their relative long analysis times. The method presented allows a fast analysis of chiral amino acids without previous time consuming chromatographic separation. The analytical measurements base on parallel kinetic resolution with pseudoenantiomeric mass tagged auxiliaries and were carried out by mass spectrometry with electrospray ionization. All 19 chiral proteinogenic amino acids were tested and Pro, Ser, Trp, His, and Glu were selected as model substrates for verification measurements. The enantiomeric excesses of amino acids with non-polar and aliphatic side chains as well as Trp and Phe (aromatic side chains) were determined with maximum deviations of the expected value less than or equal to 10ee%. Ser, Cys, His, Glu, and Asp were determined with deviations lower or equal to 14ee% and the enantiomeric excess of Tyr were calculated with 17ee% deviation. The total screening process is fully automated from the sample pretreatment to the data processing. The method presented enables fast measurement times about 1.38 min per sample and is applicable in the scope of high-throughput screenings.     

Characterization of the Tween 20-soluble membrane proteins of Acholephasma laidlawii

Methods for the purification of the four major proteins and two of the minor proteins in the Tween 20-soluble extract of A. laidlawii membranes have previously been described. The last step in the purification procedure involved an electrophoresis in a detergent-free buffer, where the concentration of Tween could be reduced by up to 2000 times. The purified proteins were found to remain soluble after removal of the bulk of the detergent. Solutions of the different protein samples contained 3 30 detergent molecules per protein molecule as determined by gas-liquid liquid chromatography. Some of the protein solutions also contained natural membrane lipids. It was probably only a fraction of detergent molecules and lipids, which was bound to the protein. Complete amino acid analysis showed that none of the proteins contained amino sugars and only one of them contained half-cystine. The specific absorbances and the molar absorption coefficients were calculated from the absorption spectra. The hydrophobicities and the partial specific volumes were calculated from the amino acid composition. The hydrophobicity values did not differ significantly from those of non-membrane proteins. Attempts to determine the sedimentation coefficients and the molecular weights were done ultracentrifugation after removal of the bulk of the detergent. The molecular weights, as determined by ultracentrifugation, were in general higher than the molecular weights determined by polyacrylamid-gel electrophoresis in sodium dodecyl sulphate (SDS), indicating that most of the proteins formed aggregates upon reducing the concentration of Tween 20. The size of these aggregates was not influenced by storage of the proteins at 0 C but it seemed to be highly affected by the speed and the time of centrifugation. The electrophoretic mobolities of the proteins were determined by free zone electrophoresis. Crossed immunoelectrophoresis was utilized to demonstrate that the Tween 20-soluble membrane proteins were not undergoing proteolysis during the preparation procedure.     

引入固氮树种对辽东落叶松人工林土壤团聚体氨基糖的影响

土壤微生物残留物是稳定性碳库的重要组分,然而固氮树种引入对落叶松人工林土壤团聚体微生物残留物分布的影响还不清楚.为了阐明固氮树种对不同团聚体粒级内微生物残留物分布的影响,本研究以氨基糖作为微生物残留物的生物标识物,比较了辽东日本落叶松人工纯林和落叶松与固氮树种赤杨混交林土壤团聚体氨基糖的分布特征.结果表明: 赤杨引入不影响团聚体氨基糖的分布,但显著提高了团聚体氨基糖含量.与纯林相比,混交林土壤不同团聚体各粒级总氨基糖含量增加1.3～1.7倍.其中,混交林土壤团聚体内总氨基糖增加量的66.5%～66.9%来自氨基葡萄糖,30.0%～30.6%来自氨基半乳糖,2.5%～3.2%来自胞壁酸.赤杨引入显著提高了>2000 μm和<250 μm团聚体中的氨基葡萄糖/胞壁酸值,但不影响250～2000 μm粒级团聚体中真菌和细菌残留物的相对贡献.此外,赤杨引入增加了土壤不同团聚体内的氨基糖对土壤有机碳的贡献,但不影响团聚体粒级之间的微生物贡献,说明赤杨对微生物贡献的影响存在空间均一性.     

Aggregability of β(1→4)-linked glucosaminoglucan originating from a sulfur-oxidizing bacterium Thiothrix nivea

ABSTRACT

β-1,4-glucosaminoglucan (GG) was prepared from the sheath of a sulfur-oxidizing bacterium Thiothrix nivea. Recently, GG was found to be adsorbed by cellulose (paper) and is therefore potentially applicable as an aminating agent for cellulose. We attempted to increase the yield of GG using a fed-batch cultivation method. Furthermore, the behavior of GG molecules in water was theoretically and experimentally investigated. NMR analysis in combination with molecular dynamics calculation suggested that GG molecules tend to form soluble aggregates in water. It was experimentally revealed that the self-aggregation is enhanced by the addition of NaCl and reduced temperature. Adsorption of GG onto cellulose via hydrogen bonding was confirmed by molecular dynamics simulation. Adsorption was also promoted in the presence of NaCl but was inhibited by a reduction in temperature. Only 11% of the amino groups in the GG-treated paper was reactive, suggesting that GG molecules adsorbed by the paper were forming aggregates.     

Number and Molecular Weights of Foot-and-Mouth Disease Virus Capsid Proteins and the Effects of Maleylation

Evidence was obtained by gel electrophoresis that foot-and-mouth disease virus (FMDV) type A(12) protein migrates mainly in a zone corresponding to polypeptide(s) approximately 25,000 daltons in molecular weight. Additional minor components were observed, four with molecular weights ranging from 10,000 to 22,500 daltons and one with a molecular weight of 37,500 daltons. The minor components comprised about 10% of the total protein and were present in variable amounts. The 75S empty capsids contained primarily 25,000-, 37,500- and 50,000-dalton zones. These molecular weights were estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate versus proteins of known molecular weight, including poliovirus and vesicular stomatitis virus proteins. Maleylation of the amino residues of FMDV protein solubilized it to about 5 to 10 mg/ml in aqueous, nondenaturing solvents. This permitted molecular weights to be estimated also by gel filtration. Maleylation of 70% of the available amino groups of the FMDV protein produced heat and sodium dodecyl sulfate-stable polymeric aggregates of 10 to 20% of the 25,000-dalton zone. It also resulted in an increase in the molecular weight of this zone by an amount equivalent (ca. 1,000) to that expected from the added maleyl residues.     

Primary structure of a protein isolated from reef shark (Carcharhinus springeri) cartilage that is similar to the mammalian C-type lectin homolog, tetranectin.

During the course of characterization of low molecular weight proteins in cartilage, we have isolated a protein from reef shark (Carcharhinus springeri) cartilage that bears a striking resemblance to the tetranectin monomer originally described by Clemmensen et al. (1986, Eur. J. Biochem. 156, 327-333). The protein was isolated by extraction of neural arch cartilage with 4 M guanidine hydrochloride, dialysis of the extract to bring the guanidine to 0.4 M (reassociating proteoglycan aggregates), followed by cesium chloride density gradient removal of the proteoglycans. The amino acid sequence had 166 amino acids and a calculated molecular weight of 18,430. The shark protein was 45% identical to human tetranectin, indicating that it was in the family of mammalian C-type lectins and that it was likely to be a shark analog of human tetranectin. The function of tetranectin is unknown; it was originally isolated by virtue of its affinity for the kringle-4 domain of plasminogen. Sequence comparison of human tetranectin and the shark-derived protein gives clues to potentially important regions of the molecule.