Positional ordering of reacting groups contributes significantly to the efficiency of proton transfer at an antibody active site

Catalytic antibody 34E4 accelerates the conversion of benzisoxazoles to salicylonitriles with surprising efficiency, exploiting a carboxylate base with an elevated pKa for proton abstraction. Mutagenesis of this antibody, produced as a chimeric Fab, confirms the prediction of a homology model that GluH50 is the essential catalytic residue. Replacement of this residue by glutamine, alanine, or glycine reduces catalytic activity by more than 2.6 x 10(4)-fold. By comparing the chemical proficiencies of the parent antibody with the chemical proficiencies of acetate and the mutants, the effective concentration of the catalytic side chain was estimated to be >51 000 M. The 2.1 kcal/mol destabilization of the transition state observed when GluH50 is replaced by aspartate suggests that positional ordering imposed by the antibody active site contributes significantly to the efficiency of proton transfer. The observation that the GluH50Ala and GluH50Gly variants could not be chemically rescued by exogenous addition of high concentrations of formate or acetate further underscores the advantage the antibody derives from covalently fixing its base at the active site. Although medium effects also play an important role in 34E4, for example in enhancing the reactivity of the carboxylate side chain through desolvation, comparison of 34E4 with less proficient antibodies shows that positioning a carboxylate in a hydrophobic binding pocket alone is insufficient for efficient general base catalysis. Our results demonstrate that structural complementarity between the antibody and its substrate in the transition state is an important and necessary component of 34E4's high activity. By harnessing an additional catalytic group that could serve as a general acid to stabilize developing negative charge in the leaving group, overall efficiencies rivaling those of highly evolved enzymes should be accessible.     

A catalytic immuno-reactor for the amperometric determination of human serum albumin

A catalytic immuno-reactor for the determination of human serum albumin (HSA) was constructed by using immobilized antibody and an amperometric detector. A sandwich assay with hemin-labeled antibody to catalyze the decomposition of hydrogen peroxide was used, the catalytic activity of the hemin-antibody conjugate being determined by measuring the decrease in hydrogen peroxide concentration. The reaction of hemin-labeled antibody with antigen was complete within 30 min and the current decrease was correlated with the HSA concentration. The relative standard deviation was about 9% at an HSA concentration of 1 mg ml^?1.     

4.50—Polarographic investigation of conformational changes of human serum albumin: Part I. Unfolding of human serum albumin by urea

The mechanism of the unfolding of human serum albumin by urea was studied using d.c. polarography. It was found that this reaction is a complex process which cannot be described in terms of a two-state transition model. As well as the Brdi?ka catalytic current we have also studied the reduction current of disulfide groups in native and denatured human serum albumin. The number of cystine residues accessible for electrode reduction in native and denatured protein was calculated. On the basis of these results a scheme for the unfolding of human serum albumin by urea is proposed.     

6,4,4'-trimethylangelicin photoadduct formation in DNA: production and characterization of a specific monoclonal antibody

The photochemical reactions of 6,4,4'-trimethylangelicin (TMA) with calf thymus DNA and an octanucleotide containing a single thymine have been characterized. HPLC analyses of enzymatically hydrolyzed TMA-DNA showed that isomeric forms of 4',5'-furan-side monoadducts were the major products. To develop monoclonal antibodies Balb c mice were immunized with the TMA-DNA complexed with methylated bovine serum albumin. The resultant antibodies were characterized by enzyme-linked immunosorbent assays (ELISA). The most sensitive antibody (7E3) has high specificity for TMA-DNA, very low cross-reactivity with DNA modified with either 4',5'-dimethylangelicin or 4'-methylangelicin and no cross-reactivity with non-modified DNA or with DNA modified with either 4'-aminomethyl-4,5',8-trimethylpsoralen or 8-methoxypsoralen. To characterize further this antibody, oligonucleotides containing specific TMA photoadducts were isolated from the photoreaction mixture by polyacrylamide gel electrophoresis and used as competitive inhibitors in the ELISA. Autoradiography of the gel showed an intense band corresponding to the 4',5'-monoadduct and two weaker unidentified bands. Antibody 7E3 reacted only with the 4',5'-monoadduct band as would be expected since this photoadduct was the principal photoadduct in the original antigen.     

QUANTUM YIELDS FOR THE CYCLIZATION AND CONFIGURATIONAL ISOMERIZATION OF 4E, 15Z-BILIRUBIN

Extraction of a solution of bilirubin configurational isomers in chloroform with an aqueous solution of human serum albumin was found to remove selectively the 4Z,15E-isomer. This phenomenon was used to develop a method for the purification of the 4E,15Z-isomer of bilirubin. The quantum yield for the cyclization and configurational isomerization of the 4E,15Z-isomer bound to a molar excess of human serum albumin was measured at 450 and 510 nm. The quantum yield for cyclization to form lumirubin was 0.12 and 0.19 at 450 and 510 nm, respectively. The quantum yield for configurational isomerization to form 4Z,15Z-bilirubin was 0.03 and 0.05 at 450 and 510 nm. An analysis of previously published data on the quantum yield for the formation of lumirubin from 4Z, 15Z-bilirubin bound to human serum albumin suggests that all of the formation of lumirubin may occur via consecutive photochemical processes with the 4E,15Z-isomer as an intermediate.     

Syntheses and characterizations of 4-(3,17beta-dihydroxyestra-1,3,5(10)-trien-6alpha- and 6beta-yl)amino-7-nitro-2,1,3-benzoxadiazoles as fluorescent probes.

4-(3,17Beta-dihydroxyestra-1,3,5(10)-trien-6alpha- and 6beta-yl)amino-7-nitro-2,1,3-benzoxadiazoles have been synthesized and characterized as fluorescent probes for use in a receptor assay and/or a homogeneous immunoassay for estradiol. The fluorescence intensities are strongly dependent upon the solvent polarity used. The intensities in water were reduced to less than 1% of those in ethyl acetate, and a blue shift was also observed in polar solvents. The quenched fluorescence in aqueous solution was recovered by adding bovine serum albumin or an anti-estradiol antibody. Adding intact estradiol inhibited the fluorescence recovered by the antibody.     

固-液相转移催化合成 VI: 醛亚胺的1,3-偶极加成反应

本文报道了用K2CO3作碱的固液相催化条件下, 醛亚胺与某些偶极试剂的1,3-偶极加成反应, 这些反应在质子性醇溶剂与K2CO3组成的两相体系中进行更为方便, 可用于吡咯烷及 唑烷衍生物的合成.     

Metal-free,noncovalent catalysis of diels-alder reactions by neutral hydrogen bond donors in organic solvents and in water

We examined the catalytic activity of substituted thioureas in a series of Diels-Alder reactions and 1,3-dipolar cycloadditions. The kinetic data reveal that the observed accelerations in the relative rates are more dependent on the thiourea substituents than on the reactants or solvent. Although the catalytic effectiveness is the strongest in noncoordinating, nonpolar solvents, such as cyclohexane, it is also present in highly coordinating polar solvents, such as water. In 1,3-dipolar cycloadditions, the thiourea catalysts demonstrate only very moderate selectivity for reactions with inverse electron demand. Our experiments emphasize that both hydrophobic and polar interactions can co-exist, making these catalysts active, even in highly coordinating solvents. This class of catalysts increases the reaction rates and endo-selectivities of Diels-Alder reactions, in a similar manner to weak Lewis acids, without concomitant product inhibition.     

Characterization of the Interaction of 6-Thioguanine with Human Serum Albumin by Surface-Enhanced Raman Scattering and Molecular Modeling

The interaction of 6-thioguanine and human serum albumin was investigated by fluorescence, ultraviolet-visible absorption, and surface-enhanced Raman scattering. The fluorescence of human serum albumin decreased with the concentration of 6-thioguanine, and the fluorescence quenching of human serum albumin by 6-thioguanine was static. Molecular modeling showed that 6-thioguanine was located in the hydrophobic cavity in subdomain IIA of human serum albumin. Surface-enhanced Raman scattering was combined with density function theory to characterize the orientation of 6-thioguanine on gold and the 6-thioguanine functional groups bonded to human serum albumin. The 6-thioguanine was shown to be tilted on the gold surface by a N-C?S moiety. The binding sites of 6-thioguanine to human serum albumin were the NH and amino groups of the pyrimidine ring of 6-thioguanine. This study may provide information regarding the metabolism of anticancer pharmaceuticals in the human body and assist in the development of effective compounds.     

Peroxidatic activity of metalloporphyrin binding to serum albumin: Enhancement effect of serum albumin on metalloporphyrin catalyzed luminol chemiluminescence reaction

The metalloporphyrin (M-P) catalyzed luminol-H₂O₂ chemiluminescence (CL) system can be significantly enhanced in the presence of serum albumins. The enhancement may be explained in terms of the formation of a molecular complex between M-P and serum albumin. The hydrophobic and coordination interactions between M-P and serum albumin were confirmed by comparing the degree of enhancement by the protein on different types of M-P catalyzed CL reactions and by comparing the catalytic activity of M-P/bovine serum albumin (BSA) and M-P/BSA/anti-BSA complexes. Only the non-ionic M-P catalyzed CL reaction was significantly enhanced, and the enhanced CL reaction was inhibited by an immunoreaction. The optimum conditions of the M-P/albumin complex catalyzed CL reaction were evaluated by using a flow-injection system, which was very similar to that using hemoglobin as the catalyst. A brief prospective on the general applicability of the enhanced CL reaction for the determination of serum albumin (2.5–500 μg/ml) is given.     

Determination of tetrahydrocannabinolic acid—carrier protein conjugate by matrix-assisted laser desorption/ionization mass spectrometry and antibody formation

In order to form the monoclonal antibody against tetrahydrocannabinolic acid (THCA), THCA was coupled with bovine serum albumin (BSA) or human serum albumin (HSA) using β-alanine as a linker or without β-alanine to produce individual antigen conjugates. The number of haptens contained in the antigen conjugates was determined by matrix-assisted laser desorption ionization mass spectrometry using BSA or HSA as an internal standard. The formation of monoclonal antibody is also discussed.     

Kinetics of the reactions of halide anions with carbocations: quantitative energy profiles for s(n)1 reactions

Rate constants for the reactions of Laser flash photolytically generated benzhydrylium ions (diarylcarbenium ions) with halide ions have been determined in various solvents, including neat and aqueous acetonitrile as well as some alcohols. Substitution of the rate constants into the correlation equation log k = s(N + E) yields the nucleophilicity parameters N for the halide ions in different solvents. Linear correlations with negative slopes are found between the nucleophilicity parameters N for Cl(-) and Br(-) in different solvents and the solvent ionizing powers Y of the corresponding solvents. Increasing halide solvation reduces the rates of carbocation/chloride combinations by approximately half as much as it increases the rates of ionizations of benzhydryl chlorides. Comparison of the solvent dependent nucleophilicity parameters N of halide anions and the nucleophilicity parameters N(1) for solvents yields a quantitative prediction of common ion rate depression, as demonstrated by the analysis of a variety of literature reported mass-law constants alpha. Combination of the rate constants for the reactions of benzhydrylium ions with halide ions (k(-)()(1)) reported in this work with the ionization constants of benzhydryl halides (k(1)) and the recently reported rate constants for the reactions of benzhydrylium ions with solvents (k(2)) yields complete quantitative free energy profiles for solvolysis reactions. The applicability of Hammond's postulate for interpreting solvolysis reactions can thus be examined quantitatively.     

Inverse electron-demand Diels-Alder (iEDDA) bioorthogonal conjugation of half-sandwich transition metallocarbonyl entities to a model protein

Novel transition metallocarbonyl complexes carrying a norbornene or an oxanorbornene group were synthesized by [4 + 2] cycloaddition between the organometallic maleimide dienophiles and cyclopentadiene or furan, respectively. The oxanorbornene adduct was obtained as a mixture of endo and exo isomers as confirmed by X-ray diffraction and NMR spectroscopy. The (oxa)norbornene groups further provided convenient chemical reporters to carry out inverse electron demand Diels-Alder (iEDDA) reactions with tetrazine derivatives. Detailed kinetic studies with a model tetrazine revealed that faster rates of reaction were determined with both isomers of the oxanorbornene complex with respect to the norbornene complexes. Eventually, incorporation of metallocarbonyl entities into bovine serum albumin equipped with tetrazine handles was achieved as shown by IR spectroscopy of the protein conjugates.     

An investigation into the role of 2,6-lutidine as an additive for the RuCl3-NaIO4 mediated oxidative cleavage of olefins to ketones

2,6-Lutidine has been identified as a beneficial additive for the oxidative cleavage of olefins to ketones by NaIO₄ in the presence of catalytic RuCl₃, improving the yield and shortening the reaction times. In the absence of 2,6-lutidine reactions stalled at the diol intermediate with incomplete conversion to the desired ketones. The reaction protocol described herein also avoids the use of harmful solvents such as CCl₄ and DCE and is tolerant of a range of functional groups.     

The high-sensitivity determination of protein concentrations by the enhancement of Rayleigh light scattering of Arsenazo-DBN

A new Rayleigh light scattering (RLS) assay of protein is presented in this paper. At the optimum pH 4.10, the weak RLS of Arsenazo-DBN can be greatly enhanced by the addition of proteins due to the interaction between protein and Arsenazo-DBN. Based on this, the reactions of Arsenazo-DBN and proteins, including bovine serum albumin, human serum album, gamma-globulin, egg albumin, lysozyme and trypsin, were studied. A new quantitative determination method for proteins has been developed. The linear range for human serum albumin, for example, is 0.085-34.62 micrograms mL-1 with a detection limit of 44.8 ng mL-1. Besides high sensitivity, the method is characterized by good reproducibility, rapidity of reaction, good stability, and few interfering substances. The determination of the proteins in human serum and urine samples by this method give results very close to those obtained using Coomassie Brilliant Blue G-250 colorimetry, with relative standard deviations of 0.7-2.5%.     

Highly efficient antibody-catalyzed deuteration of carbonyl compounds

Antibody 38C2 efficiently catalyzes deuterium-exchange reactions at the alpha position of a variety of ketones and aldehydes, including substrates that have a variety of sensitive functional groups. In addition to the regio- and chemoselectivity of these reactions, the catalytic rates (kcat) and rate-enhancement values (kcat/kun) are among the highest values ever observed with catalytic antibodies. Comparison of the substrate range of the catalytic antibody with highly evolved aldolase enzymes, such as rabbit-muscle aldolase, highlights the much broader practical scope of the antibody, which accepts a wide range of substrates. The hydrogen-exchange reaction was used for calibration and mapping of the antibody active site. Isotope-exchange experiments with cycloheptanone reveal that the formation of the Schiff base species (as concluded from the 16O/18O exchange rate at the carbonyl oxygen) is much faster than the formation of the enamine intermediate (as concluded from the H/D exchange rate), and both steps are faster than the antibody-catalyzed aldol addition reaction.     

A catalytic antibody against a tocopherol cyclase inhibitor

The cyclic ammonium cation 5 and its guanidinium analogue 4 are inhibitors of tocopherol cyclase. Monoclonal antibodies were raised against protein conjugates of the haptens 1-3 and screened for catalytic reactions with alkene 8, a short chain analogue of the natural substrate phytyl-hydroquinone 6, and its enol ether analogues 10a,b. Antibody 16E7 raised against hapten 3 was found to catalyze the hydrolysis of Z enol ether 10a to form hemiacetal 12 with an apparent rate acceleration of k(cat)/k(uncat)=1400. Antibody 16E7 also catalyzed the elimination of Kemp's benzisoxazole 59. The absence of cyclization in the reaction of enol ether 10a was attributed to the competition of water molecules for the oxocarbonium cation intermediate within the antibody binding pocket. Hapten and reaction design features contributing to this outcome are discussed. Antibody 16E7 provides the first example of a carboxyl group acting both as an acid in an intrinsically acid-catalyzed process and as a base in an intrinsically base-catalyzed process, as expected from first principles. In contrast to the many examples of general-acid-catalyzed processes known to be catalyzed by catalytic antibodies, the specific-acid-catalyzed cyclization of phytyl-hydroquinone 6 or its analogue 8 still eludes antibody catalysis.     

凝胶电泳-激光烧蚀进样电感耦合等离子体质谱与非特异性同位素稀释法联用技术定量分析人血清中蛋白

建立了聚丙烯酰胺凝胶电泳(PAGE)-激光烧蚀进样电感耦合等离子体质谱(LA-ICP-MS)与非特异性同位素稀释法联用技术, 通过测定蛋白条带上硫元素的含量, 实现了人血清中蛋白的定量分析. 血清电泳分离条件为: 分离胶浓度(质量分数)为7.5%, 浓缩胶浓度为4%, 电泳电压100 V. 为优化激光烧蚀实验条件, 以LA-ICP-MS测得不同浓度硫富集凝胶中³²S的信号强度, 在1~400 μg/g范围内呈现良好的线性关系, R²=0.993. 采用外标法对血清中的转铁蛋白和白蛋白进行了相对定量分析. 此外, 重点采用“电泳后同位素稀释”方法实现了蛋白条带上³⁴S稀释剂的加入, 对血清中转铁蛋白和白蛋白进行了绝对定量分析; 采用人血清蛋白标准物质ERM-DA470/IFCC对建立的方法体系进行了验证, 结果表明2种蛋白的定量结果与标准值吻合, 方法的准确性好, 且测量精度优于外标法.     

A novel approach for a non competitive capillary electrophoresis immunoassay with laser-induced fluorescence detection for the determination of human serum albumin

A new non competitive capillary electrophoresis immunoassay format based on a separation into a capillary modified by analyte immobilisation is described. The injection of an excess of labelled antibody off-line preincubated with the analyte allows the surface capture of the free antibody and the immunocomplex detection. It was developed using the human serum albumin (HSA) as analyte, two FITC-labelled antibodies and a HSA covalently linked capillary. Two calibration curves with good run-to-run reproducibility and LOD--respectively 14.0 nM for the FITC-polyclonal antibody and 9.0 nM for the FITC-monoclonal antibody--were achieved. The assay was applied to HSA determination in spiked samples of human urine with acceptable recoveries.     

Bilirubin/rat serum albumin interaction

Essential differences are demonstrated between bilirubin binding to rat serum proteins and to albumin in human serum. Acidimetric titration of rat serum with and without added bilirubin shows that binding of bilirubin acid in the range of pH from 6.8 to 8.8 takes place with release of less than one hydrogen ion per molecule of bound bilirubin. With human serum, two hydrogen ions are released, indicating binding of bilirubin dianion. The binding equilibrium of N-[4-[(4-aminophenyl)-sulfonyl]phenyl]-acetamide (MADDS) to rat serum albumin is influenced slightly by cobinding of bilirubin whereas MADDS and bilirubin bind competitively to human serum albumin. Finally, the rate of oxidation of bilirubin with hydrogen peroxide and peroxidase is decreased moderately by addition of rat serum albumin and strongly by the human protein, indicating that biliribin in its complex with rat serum albumin is subject to oxidation while the complex with human serum albumin is protected. These differences should be considered when rats are used as a model in experimental studies aiming at prevention of bilirubin encephalopathy in human neonates.