Expression of the catalytic antibodies in eukaryotic systems

Expression of recombinant antibodies in mammalian cells is one of key problems in immunobiotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively done in yeast cells. We obtained expression strains of the methylotrophic beast Pichia pastoris producing single chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab) and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (Phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and single chain antibody A.17 expressed in CHO and E. coli cells respectively.     

Expression of catalytic antibodies in eukaryotic systems

Expression of recombinant antibodies in mammalian cells is one of the key problems in immuno-biotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively performed in yeast cells. We obtained expression strains of the methylotrophic yeast Pichia pastoris producing single-chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab), and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant K _d and the first order constant (k ₂) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and to the single-chain antibody A.17 expressed in CHO and E. coli cells, respectively.     

Crystal structure of the broadly cross-reactive HIV-1-neutralizing Fab X5 and fine mapping of its epitope

The human monoclonal antibody Fab X5 neutralizes a broad range of HIV-1 primary isolates. The crystal structure of X5 has been determined at 1.9 A resolution. There are two crystallographically independent Fab fragments in the asymmetric unit. The crystallographic R value for the final model is 0.22. The antibody-combining site features a long (22 amino acid residues) CDR H3 with a protruding hook-shaped motif. The X5 structure and site-directed mutagenesis data suggest that X5 amino acid residues W100 and Y100F in the CDR H3 motif may be critical for the binding of Fab X5 to gp120. X5 bound to a complex of a CD4 mimetic and gp120 with approximately the same kinetics and affinity as to a CD4-gp120 complex, suggesting that specific interactions between CD4 and X5 are unlikely to contribute to the binding of X5 to gp120-CD4 complexes. Binding of X5 to alanine scanning mutants of gp120JR-CSF complexed with CD4 suggested a critical role of the highly conserved amino acid residues at positions 423 and 432. The X5 structure and fine mapping of its epitope may assist in the elucidation of the mechanisms of viral entry and neutralization, and the development of HIV-1 inhibitors and vaccines.     

Dissection of the ATP-Dependent Conformational Change Cycle of a Group II Chaperonin

Group II chaperonin captures an unfolded protein while in its open conformation and then mediates the folding of the protein during ATP-driven conformational change cycle. In this study, we performed kinetic analyses of the group II chaperonin from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TKS1-Cpn), by stopped-flow fluorometry and stopped-flow small-angle X-ray scattering to reveal the reaction cycle. Two TKS1-Cpn variants containing a Trp residue at position 265 or position 56 exhibit nearly the same fluorescence kinetics induced by rapid mixing with ATP. Fluorescence started to increase immediately after the start of mixing and reached a maximum at 1–2 s after mixing. Only in the presence of K⁺ that a gradual decrease in fluorescence was observed after the initial peak. Similar results were obtained by stopped-flow small-angle X-ray scattering. A rapid fluorescence increase, which reflects nucleotide binding, was observed for the mutant containing a Trp residue near the ATP binding site (K485W), irrespective of the presence or absence of K⁺. Without K⁺, a small, rapid fluorescence decrease followed the initial increase, and then a gradual decrease was observed. In contrast, with K⁺, a large, rapid fluorescence decrease occurred just after the initial increase, and then the fluorescence gradually increased. Finally, we observed ATP binding signal and also subtle conformational change in an ATPase-deficient mutant with K485W mutation. Based on these results, we propose a reaction cycle model for group II chaperonins.     

Structural basis for antibody catalysis of a disfavored ring closure reaction.

The catalysis of disfavored chemical reactions, especially those with no known natural enzyme counterparts, is one of the most promising achievements of catalytic antibody research. Antibodies 5C8, 14B9, 17F6, and 26D9, elicited by two different transition-state analogues, catalyze disfavored endo-tet cyclization reactions of trans-epoxy alcohols, in formal violation of Baldwin's rules for ring closure. Thus far, neither chemical nor enzyme catalysis has been capable of emulating the extraordinary activity and specificity of these antibodies. X-ray structures of two complexes of Fab 5C8 with the original hapten and with an inhibitor have been determined to 2.0 A resolution. The Fab structure has an active site that contains a putative catalytic diad, consisting of AspH95 and HisL89, capable of general acid/base catalysis. The stabilization of a positive charge that develops along the reaction coordinate appears to be an important factor for rate enhancement and for directing the reaction along the otherwise disfavored pathway. Sequence analysis of the four catalytic antibodies, as well as four inactive antibodies that strongly bind the transition-state analogues, suggests a conserved catalytic mechanism. The occurrence of the putative base HisL89 in all active antibodies, its absence in three out of the four analyzed inactive antibodies, and the rarity of a histidine at this position in immunoglobulins support an important catalytic role for this residue.     

A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli

1. Benzyl phosphonates were prepared and their potentialities as chromophoric reagents for the exploration of the substrate-binding site of Escherichia coli alkaline phosphatase were investigated. 4-Nitrobenzylphosphonate is a competitive inhibitor of the enzyme. 2-Hydroxy-5-nitrobenzylphosphonate changes its spectrum on binding to the enzyme. This spectral change is reversed when the phosphonate is displaced from the enzyme by substrate. 2. The kinetics of the reaction of 2-hydroxy-5-nitrophenylphosphonate were studied by the stopped-flow and the temperature-jump techniques. It was found that the combination of the phosphonate with the enzyme occurred in two successive and reversible steps: enzyme-phosphonate complex-formation followed by rearrangement of the complex. The spectral change is associated with the rearrangement. At pH8 in 1m-sodium chloride at 22 degrees the rate constant is 167sec.(-1) for the rearrangement of the initially formed binary complex and is 18sec.(-1) for the reverse process. 3. It has previously been proposed that the reactions of phosphatase with its substrates include a distinct step between enzyme-substrate combination and chemical catalysis. The rate constant involved could be predicted but not measured from experiments with substrates. The value for the rate constant measured from the rate of the enzyme-phosphonate rearrangement is in excellent agreement with the predicted value. A model for the reaction mechanism is proposed that includes a conformation change in response to phosphate ester binding before phosphate transfer from substrate to enzyme.     

Directed evolution of an anti-human red blood cell antibody

We have earlier described a haemagglutination-based assay for on-site detection of antibodies to HIV using whole blood. The reagent in this assay comprises of monovalent Fab fragment of an anti-human RBC antibody fused to immunodominant antigens of HIV-1 and HIV-2. In the present work, we describe a rational and systematic method for directed evolution of scFv and Fab antihuman RBC antibody fragments. Based on homology modeling and germline sequence alignments of antibodies, target residues in the anti-RBC MAb B6 sequence were identified for mutagenesis. A combinatorial library of 10⁷ clones was constructed and subjected to selection on whole RBC under competitive binding conditions to identify several phage-displayed B6 scFv clones with improved binding as determined in an agglutination assay. Selected V_L and V_H sequences were shuffled to generate a second generation phage-displayed Fab library which on panning yielded Fab clones with several fold better binding than wild type. The mutants with better binding also displayed more Fab molecules per phage particle indicating improved in vivo folding which was also confirmed by their increased periplasmic secretion compared to the wild type. The mutant Fab molecules also showed superior characteristics in large scale production by in vitro folding of LC and Fd. The biophysical measurements involving thermal and chemical denaturation and renaturation kinetics clearly showed that two of the mutant Fab molecules possessed significantly improved characteristics as compared to the wild type B6 Fab. Structural modelling revealed that B6 Fab mutants had increased hydrogen bonding resulting in increased stability. Our approach provides a novel and useful strategy to obtain recombinant antibodies with improved characteristics.Key words: phage display, antibody maturation, Fab, antibody folding, scFv, agglutination     

Addition of oxygen to the diiron(II/II) cluster is the slowest step in formation of the tyrosyl radical in the W103Y variant of ribonucleotide reductase protein R2 from mouse

Activation of O2 by the diiron(II/II) cluster in protein R2 of class I ribonucleotide reductase generates the enzyme's essential tyrosyl radical. A crucial step in this reaction is the transfer of an electron from solution to a diiron(II/II)-O2 adduct during formation of the radical-generating, diiron(III/IV) intermediate X. In the reaction of R2 from Escherichia coli, this electron injection is initiated by the rapid (>400 s-1 at 5 degrees C), transient oxidation of the near-surface residue, tryptophan 48, to a cation radical and is blocked by substitution of W48 with F, A, G, Y, L, or Q. By contrast, a study of the cognate reaction in protein R2 from mouse suggested that electron injection might be the slowest step in generation of its tyrosyl radical, Y177* [Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gr?slund, A. (1998) J. Biol. Chem. 273, 21463-21472]. The crucial evidence was the observation that Y177* production is slowed by approximately 30-fold upon substitution of W103, the cognate of the electron-shuttling W48 in E. coli R2, with tyrosine. In this work, we have applied stopped-flow absorption and freeze-quench electron paramagnetic resonance and M?ssbauer spectroscopies to the mouse R2 reaction to evaluate the possibility that an already sluggish electron-transfer step is slowed by 30-fold by substitution of this key residue. The drastically reduced accumulation of cluster X, failure of precursors to the intermediate to accumulate, and, most importantly, first-order dependence of the rate of Y177* formation on the concentration of O2 prove that addition of O2 to the diiron(II/II) cluster, rather than electron injection, is the slowest step in the R2-W103Y reaction. This finding indicates that the basis for the slowing of Y177* formation by the W103Y substitution is an unexpected secondary effect on the structure or dynamics of the protein, its diiron(II/II) cluster, or both rather than the expected chemical effect on the electron injection step.     

Probing the dynamics of a mobile loop above the active site of L1, a metallo-beta-lactamase from Stenotrophomonas maltophilia, via site-directed mutagenesis and stopped-flow fluorescence spectroscopy

A structural feature shared by the metallo-beta-lactamases is a flexible loop of amino acids that extends over their active sites and that has been proposed to move during the catalytic cycle of the enzymes, clamping down on substrate. To probe the movement of this loop (residues 152-164), a site-directed mutant of metallo-beta-lactamase L1 was engineered that contained a Trp residue on the loop to serve as a fluorescent probe. It was necessary first, however, to evaluate the contribution of each native Trp residue to the fluorescence changes observed during the catalytic cycle of wild-type L1. Five site-directed mutants of L1 (W39F, W53F, W204F, W206F, and W269F) were prepared and characterized using metal analyses, CD spectroscopy, steady-state kinetics, stopped-flow fluorescence, and fluorescence titrations. All mutants retained the wild-type tertiary structure and bound Zn(II) at levels comparable with wild type and exhibited only slight (<10-fold) decreases in k(cat) values as compared with wild-type L1 for all substrates tested. Fluorescence studies revealed a single mutant, W39F, to be void of the fluorescence changes observed with wild-type L1 during substrate binding and catalysis. Using W39F as a template, a Trp residue was added to the flexile loop over the active site of L1, to generate the double mutant, W39F/D160W. This double mutant retained all the structural and kinetic characteristics of wild-type L1. Stopped-flow fluorescence and rapid-scanning UV-visible studies revealed the motion of the loop (k(obs) = 27 +/- 2 s(-1)) to be similar to the formation rate of a reaction intermediate (k(obs) = 25 +/- 2 s(-1)).     

Role of active site residues and solvent in proton transfer and the modulation of flavin reduction potential in bacterial morphinone reductase

The reactions of several active site mutant forms of bacterial morphinone reductase (MR) with NADH and 2-cyclohexen-1-one as substrates have been studied by stopped-flow and steady-state kinetic methods and redox potentiometry. The enzymes were designed to (i) probe a role for potential proton donors (Tyr-72 and Tyr-356) in the oxidative half-reaction of MR; (ii) assess the function of a highly conserved tryptophan residue (Trp-106) in catalysis; (iii) investigate the role of Thr-32 in modulating the FMN reduction potential and catalysis. The Y72F and Y356F enzymes retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key proton donors in the oxidative reaction of MR. Taken together with our recently published data (Messiha, H. L., Munro, A. W., Bruce, N. C., Barsukov, I., and Scrutton, N. S. (2005) J. Biol. Chem. 280, 4627-4631) that rule out roles for Cys-191 (corresponding with the proton donor, Tyr-196, in the structurally related OYE1 enzyme) and His-186 as proton donors, we infer solvent is the source of the proton in the oxidative half-reaction of MR. We demonstrate a key role for Thr-32 in modulating the reduction potential of the FMN, which is decreased approximately 50 mV in the T32A mutant MR. This effects a change in rate-limiting step in the catalytic cycle of the T32A enzyme with the oxidizing substrate 2-cyclohexenone. Despite the conservation of Trp-106 throughout the OYE family, we show this residue does not play a major role in catalysis, although affects on substrate and coenzyme binding are observed in a W106F enzyme. Our studies show some similarities, but also major differences, in the catalytic mechanism of MR and OYE1, and emphasize the need for caution in inferring mechanism by structural comparison of highly related enzymes in the absence of solution studies.     

Docking of a human rhinovirus neutralizing antibody onto the viral capsid

The structure of the complex between the Fab fragment of a human rhinovirus serotype 2 (HRV2) neutralizing antibody (8F5) and a cross-reactive synthetic peptide derived from the viral capsid protein VP2 has been recently determined by crystallographic methods.¹ The conformation adopted by the peptide was very similar to and could be superimposed onto the corresponding region of the viral protein VP2 of human rhinovirus 1A (HRV1A) whose three-dimensional structure is known.² The structure of the Fab fragment determined in the complex was docked onto the viral capsid using the superimposition transformation found for the peptide. In the resulting model the Fab protrudes almost radially to about 60 Å from the surface of the virion without any major steric problem. The Fab fragment was then placed on each one of the 60 equivalent epitopes using the T = 1 icosahedral symmetry of the virus. The closest pairs of Fab fragments are related by viral 2-fold axes and run almost parallel to each other without clashing. These axes of symmetry from the viral particle could thus be coincident with the dyad axes of the antibodies. Furthermore, comparison of the three-dimensional structure of the Fab/peptide complex with the structure of the Fab fragment alone³ indicates that the flexibility of the antibody's elbow would facilitate bivalent attachment to the same viral particle. In accordance with the docking results, experimental determination of the stoichiometry of binding yielded a ratio of 30 IgG molecules per virion also suggesting bivalent attachment of antibody 8F5 onto the viral particle. The neutralization of viral infectivity, being neither aggregation (this paper) nor inhibition of receptor binding,⁴ might be mainly achieved by reducing viral spread from cell to cell and/or inhibition of uncoating. © 1995 Wiley-Liss, Inc.     

Structural basis of the transition-state stabilization in antibody-catalyzed hydrolysis

The catalytic antibody 6D9, which was raised against a transition-state analogue (TSA), catalyzes the hydrolysis of a non-bioactive chloramphenicol monoester to generate chloramphenicol. It has been shown that 6D9 utilizes the binding affinity in the catalysis; the differential affinity of the TSA relative to the substrate is equal to the rate enhancement. To reveal the recognition mechanism of 6D9 for the TSA and the substrate, we performed NMR analysis of the Fv fragment of 6D9 (6D9-Fv), together with site-directed mutagenesis and stopped-flow kinetic analyses. Among six 6D9-Fv mutants, Y58(H)A and W100i(H)A displayed significant reductions in their affinities to the TSA, while their substrate-binding affinities were identical with that of the wild-type 6D9-Fv. The stopped-flow kinetic studies revealed that the TSA binding to 6D9-Fv occurred by an induced-fit mechanism. In contrast, no induced-fit type of TSA-binding mechanism was observed for Y58(H)A and W100i(H)A. From NMR experiments, we identified the residues with chemical shifts that were perturbed by the ligand-binding. The residues affected by the TSA binding were located on the TSA-binding site determined by the X-ray study, and on the regions far from the binding site. On the other hand, the residues affected by the substrate binding were localized on the TSA-binding site. As for W100i(H)A, no residue other than those in the binding site was affected by the ligand binding. On the basis of these results and the crystal structure, we concluded that the TSA binding induced a conformational change involving the formation of aromatic-aromatic interactions and a hydrogen bond. These interactions can account for the differential affinity for the TSA relative to the substrate. W100i(H) probably plays an important role in inducing the conformational changes. The present NMR studies have enabled us to visualize the concept of transition-state stabilization in enzymatic catalysis, in which the transition-state contacts are better than those of the substrate.     

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody–hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen‐binding fragment (Fab) derived from the TN1 antibody (TN1‐Fab). To clarify the mechanism by which hTPO is recognized by TN1‐Fab the conformation of free TN1‐Fab was determined to a resolution of 2.0 Å using X‐ray crystallography and compared with the hTPO‐bound form of TN1‐Fab determined by a previous study. This structural comparison revealed that the conformation of TN1‐Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen‐binding site (paratope) of TN1‐Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (?1.52 ± 0.05 kJ mol^?1 K^?1) differed significantly from calculations based upon the X‐ray structure data of the hTPO‐bound and unbound forms of TN1‐Fab (?1.02 ～ 0.25 kJ mol^?1 K^?1) suggesting that hTPO undergoes an induced‐fit conformational change combined with significant desolvation upon TN1‐Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.     

A Strain-Specific Epitope of Enterovirus 71 Identified by Cryo-Electron Microscopy of the Complex with Fab from Neutralizing Antibody

Enterovirus 71 (EV71) is a picornavirus that causes outbreaks of hand, foot, and mouth disease (HFMD), primarily in the Asia-Pacific area. Unlike coxsackievirus A16, which also causes HFMD, EV71 induces severe neuropathology leading to high fatalities, especially among children under the age of 6 years. Currently, no established vaccines or treatments are available against EV71 infection. The monoclonal antibody MA28-7 neutralizes only specific strains of EV71 that have a conserved glycine at amino acid VP1-145, a surface-exposed residue that maps to the 5-fold vertex and that has been implicated in receptor binding. The cryo-electron microscopy structure of a complex between EV71 and the Fab fragment of MA28-7 shows that only one Fab fragment occupies each 5-fold vertex. A positively charged patch, which has also been implicated in receptor binding, lies within the Fab footprint. We identify the strain-specific epitope of EV71 and discuss the possible neutralization mechanisms of the antibody.     

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL,FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES*

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1–3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro¹⁰³. A molecular dynamics simulation and mutational analyses revealed that Arg⁴⁰ in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.     

Bidirectional electron transfer in photosystem I: replacement of the symmetry-breaking tryptophan close to the PsaB-bound phylloquinone A1B with a glycine residue alters the redox properties of A1B and blocks forward electron transfer at cryogenic temperatures

A conserved tryptophan residue located between the A(1B) and F(X) redox centres on the PsaB side of the Photosystem I reaction centre has been mutated to a glycine in Chlamydomonas reinhardtii, thereby matching the conserved residue found in the equivalent position on the PsaA side. This mutant (PsaB:W669G) was studied using EPR spectroscopy with a view to understanding the molecular basis of the reported kinetic differences in forward electron transfer from the A(1A) and the A(1B) phyllo(semi)quinones. The kinetics of A(1)(-) reoxidation due to forward electron transfer or charge recombination were measured by electron spin echo spectroscopy at 265 K and 100 K, respectively. At 265 K, the reoxidation kinetics are considerably lengthened in the mutant in comparison to the wild-type. Under conditions in which F(X) is initially oxidised the kinetics of charge recombination at 100 K are found to be biphasic in the mutant while they are substantially monophasic in the wild-type. Pre-reduction of F(X) leads to biphasic kinetics in the wild-type, but does not alter the already biphasic kinetic properties of the PsaB:W669G mutant. Reduction of the [4Fe-4S] clusters F(A) and F(B) by illumination at 15 K is suppressed in the mutant. The results provide further support for the bi-directional model of electron transfer in Photosystem I of C. reinhardtii, and indicate that the replacement of the tryptophan residue with glycine mainly affects the redox properties of the PsaB bound phylloquinone A(1B).     

The Principal Neutralizing Determinant of HIV-1IIIB: Conformation of the Peptide Bound to a Neutralizing Antibody Studied by 2D-NMR

Summary RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5β raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser⁶-Thr¹⁹ are part of the epitope, while Lys⁵ and Ile²⁰ are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel β-strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5β.     

用硫氰酸盐洗脱法直接测定噬菌体抗体的相对亲和力

抗体与相应抗原的结合可以被硫氰酸盐洗脱而解离,抗体的亲和力越高则解离所需要的硫氰酸盐浓度就越大,这一原理在传统的免疫学实验中被用来测定单克隆抗体或多克隆抗体的相对亲和力。如果证明该原理同样适用于噬菌体抗体库技术,则可以建立一种直接测定噬菌体抗体相对亲和力的简便方法。首先将噬菌体抗体与工作浓度的硫氰酸盐共孵育,以证明这一过程并不影响其后的ELISA反应,然后参照硫氰酸盐洗脱法测定完整抗体分子和Fab段相对亲和力的方法,在ELISA实验中以酶标抗M13为二抗检测了5个单克隆噬菌体抗体的相对亲和力,并与相对应的可溶性Fab段的相对亲和力进行了比较。被测抗体中包括3个克隆的抗角蛋白抗体和2个克隆的抗乙型肝炎表面抗原抗体。结果发现,用硫氰酸盐洗脱法测定5个噬菌体抗体所得到的亲和力排序与测定其相应可溶性Fab段所得结果一致,表明硫氰酸盐洗脱法可作为一种简便快速的方法用来直接测定噬菌体抗体的相对亲和力。     

The reaction of hemin with H2O2

The kinetics of formation of the dominant intermediate (CII) formed between hemin and H2O2 has been studied by the stopped-flow method. CII is preceded by a precursor (CI) for which a steady state is established at an early stage of the reaction. The formation of CI from hemin and H2O2 causes only a marginal change in the optical absorbance (A). The transition CI----CII is accompanied by a substantial decrease of A in the Soret region. Relevant rate constants (or combinations of them) and the molar absorption coefficients of the intermediates at 400 nm have been determined. The absorption spectrum of CII in the Soret region has been evaluated. Aspects of the catalysis of decomposition of H2O2 by hemin in relation to the Fe3+ ion and catalase are discussed.     

Nuclear magnetic resonance evidence for a structural intermediate at an early stage in the refolding of ribonuclease A

The kinetics of refolding of heat-unfolded ribonuclease A have been studied by Fourier transform proton nuclear magnetic resonance at 10 °C, pH 2. A single refolding reaction is observed: it corresponds to the slow-refolding reaction seen in stopped-flow studies of refolding at higher temperatures. There are two results of interest for the mechanism of protein folding. (1) A new resonance (X) is observed that shows the presence of a structural intermediate in refolding. (2) The α-helix close to the N-terminal end of ribonuclease A apparently forms rapidly when the unfolded protein is brought to refolding conditions.The folding intermediate has been studied by monitoring the C-2 protons of the four histidine residues. The intermediate contains one residue (X) in a partly folded environment and the other three residues in unfolded environments. The composite resonance (U) of these three protons at 10 °C agrees with the average chemical shift of the histidine residues in heat-unfolded ribonuclease A at high temperatures. During refolding at 10 °C, the resonance intensities of U and X disappear at the same rate that the spectrum of native ribonuclease A is regained.Partial deuteration experiments show that X is either histidine 12 or 119. Comparative studies of the amino-terminal fragment 1–20 of ribonuclease A indicate that X is histidine 12. The appearance of structure in this peptide can be followed by temperature-dependent changes in the chemical shift of histidine 12. At 10 °C the chemical shifts of histidine 12 and X agree closely. These results are consistent with the circular dichroism study of peptide 1–13 by Brown &; Klec (1971), who concluded that helix formation occurs at low temperatures.