Quaternary association and reactivation of dimeric concanavalin A

The reconstitution of dimeric concanavalin A (ConA) in terms of quaternary association and reactivation, after denaturation in urea, has been investigated using intrinsic fluorescence, 8-anilino-1-naphthalenesulfonate (ANS) binding, far-UV circular dichroism (CD), and an activity assay developed through a combination of affinity binding and the o-phthalaldehyde (OPA) procedure of protein estimation. The equilibrium denaturation of dimeric ConA in urea exhibits a biphasic unfolding pathway involving an intermediate with hydrophobic exposure, and the overall free energy of stabilization for the dimeric protein is obtained as 16.3 kcal mol(-1). The time course of reassociation and regain of activity during reconstitution reveals that the reactivation of ConA runs almost parallel to the process of subunit association. The reactivation reaction follows second-order kinetics, with a rate constant (k) of 2.6 x 10(2) M(-1) s(-1). These results may provide insight into the relationship between quaternary association and function of legume lectins.     

Effect of coenzymes and temperature on the process of in vitro refolding and reassociation of lactic dehydrogenase isoenzymes.

Dissociation and deactivation of the H4 and M4 isoenzymes of lactic dehydrogenase in strong denaturants may be reversed with a yield of reactivation up to 100%. The products of reconstitution are indistinguishable from the native enzymes as far as the Michaelis constants and the dissociation constants for substrate and coenzyme as well as spectral and hydrodynamic properties are concerned. The presence of NAD+ and NADH does not affect either the conformational state of the product of reconstitution, or the kinetics of reactivation, using the pure apoenzymes as a reference. At 20 degrees C the kinetics of reactivation for LDH-M4 in the presence and absence of coenzyme may be quantitatively described by a second-order rate equation (k2 = 23.4 +/- 2.6 mM-1S-1) while LDH-H4 is characterized by a uni-bimolecular reaction sequence (k1 = 1.45 +/- 0.45 X 10(-3)-S-1, k2 = 5 +/- 1 mM-1S-1), in agreement with earlier observations (Rudolph, R., et al. (1977), Biochemistry 16, 3384-3390). Regarding the influence of temperature on the rate of reactivation no significant anomalies are detectable within the range of 0-25 degrees C. The (apparent) activation energies, taken from the linear Arrhenius plots, are 58 kcal/mol for the association reaction of LDH-M4, and 41 kcal/mol for the transconformation reaction of LDH-H4.     

Reassociation of dimeric cytoplasmic malate dehydrogenase is determined by slow and very slow folding reactions

Malate dehydrogenase occurs in virtually all eucaryotic cells in mitochondrial and cytoplasmic forms, both of which are composed of two identical subunits. The reactivation of the mitochondrial isoenzyme has been the subject of previous studies [Jaenicke, R., Rudolph, R., & Heider, I. (1979) Biochemistry 18, 1217-1223]. In the present study, the reconstitution of cytoplasmic malate dehydrogenase from porcine heart after denaturation by guanidine hydrochloride has been determined. The enzyme is denatured by greater than 1.2 M guanidine hydrochloride; upon reconstitution, approximately 60% of the initial native enzyme can be recovered. The kinetics of reconstitution after maximum unfolding by 6 M guanidine hydrochloride were analyzed by fluorescence, far-ultraviolet circular dichroism, chemical cross-linking with glutaraldehyde, and activity measurements. After fast folding into structured intermediates (less than 1 min), formation of native enzyme is governed by two parallel slow and very slow first-order folding reactions (k1 = 1.3 X 10(-3) S-1 and k2 = 7 X 10(-5) S-1 at 20 degrees C). The rate constant of the association step following the slow folding reaction (determined by k1) must be greater than 10(6) M-1 S-1. The energy of activation of the slow folding step is of the order of 9 +/- 1 kcal/mol; the apparent rate constant of the parallel very slow folding reaction is virtually temperature independent. The intermediates of reassociation must be enzymatically inactive, since reactivation strictly parallels the formation of native dimers. Upon acid dissociation (pH 2.3), approximately 35% of the native helicity is preserved, as determined by circular dichroism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cystine-mediated oligomerization of the Atlantic salmon serum C-type lectin

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a C-type lectin that binds to bacteria including salmon pathogens. SSL has been shown to be oligomeric in salmon serum and it displays a stoichiometric band-laddering pattern when analyzed by SDS-PAGE under non-reducing conditions. In this study, a model was generated for SSL isoform 2 in silico in order to identify cysteines that are available to form intermolecular disulfide bonds facilitating oligomerization. Then, recombinant SSL was expressed in E. coli and mutants were produced at positions Cys72 and Cys149. The SSL preparations were purified by metal-affinity chromatography and shown to be functional by carbohydrate-affinity chromatography. The recombinant SSL formed oligomers, which were evident by non-reducing covalent cross-linking and non-reducing SDS-PAGE; however, the band patterns were different for the mutants, with the maximal and predominant multimer sizes distinct from the wild-type recombinant lectin. Further examination of oligomerization by size exclusion chromatography revealed a subunit number from 35 to at least 110 for the wild-type recombinant SSL and subunit numbers below 9 for each mutant SSL oligomer. Thus, both cysteines were found to contribute to oligomerization of SSL.     

Visualization of galectin-3 oligomerization on the surface of neutrophils and endothelial cells using fluorescence resonance energy transfer

Galectin-3, a member of the galectin family of carbohydrate binding proteins, is widely expressed, particularly in cells involved in the immune response. Galectin-3 has also been indicated to play a role in various biological activities ranging from cell repression to cell activation and adhesion and has, thus, been recognized as an immunomodulator. Whereas those activities are likely to be associated with ligand cross-linking by this lectin, galectin-3, unlike other members of the galectin family, exists as a monomer. It has consequently been proposed that oligomerization of the N-terminal domains of galectin-3 molecules, after ligand binding by the C-terminal domain, is responsible for this cross-linking. The oligomerization status of galectin-3 could, thus, control the majority of its extracellular activities. However, little is known about the actual mode of action through which galectin-3 exerts its function. In this report we present data suggesting that oligomerization of galectin-3 molecules occurs on cell surfaces with physiological concentrations of the lectin. Using galectin-3 labeled at the C terminus with Alexa 488 or Alexa 555, the oligomerization between galectin-3 molecules on cell surfaces was detected using fluorescence resonance energy transfer. We observed this fluorescence resonance energy transfer signal in different biological settings representing the different modes of action of galectin-3 that we previously proposed; that is, ligand crosslinking leading to cell activation, cell-cell interaction/adhesion, and lattice formation. Furthermore, our data suggest that galectin-3 lattices are robust and could, thus, be involved, as previously proposed, in the restriction of receptor clustering.     

Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein

The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.     

Reaction rates for the production of selected hormones by solid-phase synthesis.

Reaction rates have been measured for the production of selected bradykinins, angiotensins, and endorphins by solid-phase synthesis. Reactor liquid concentrations are monitored by using a UV detector and flow cell. Experimentation has been limited to low excesses of the t-Boc amino symmetrical anhydride. More than 500 attachments have been monitored. Most data obtained from resins with 1% cross-linking show second-order behavior with reaction rate constants between 0.5 and 8 L/(mol.s). The reaction rate is affected by chemical structures of both the attached amino symmetrical anhydride and the anchored amino terminus on the peptide fragment. Increasing reaction temperature and initial solution concentrations promotes reaction rate. The structure of the polymer support affects not only the reaction rate but also the observed reaction kinetics.     

Signal transmission by epidermal growth factor receptor: coincidence of activation and dimerization.

Dimerization of epidermal growth factor receptor dissolved in a solution of nonionic detergent was followed with a resolution of 1 min by quantitative cross-linking with glutaraldehyde. Upon addition of epidermal growth factor to the solution, the initially monomeric protein dimerized in a reaction that was second-order in the concentration of receptor. A second-order rate constant, on the basis of enzymatic activity as a measure of the concentration of functional receptor, was calculated from time courses of dimerization at various initial concentrations of receptor. The activation of the protein tyrosine kinase of the receptor was monitored directly under the same conditions with an exogenous substrate. The increase in tyrosine kinase activity displayed kinetics that were also second-order in the concentration of receptor. A second-order rate constant for the activation of the tyrosine kinase could be calculated from the time courses. The second-order rate constant for the activation of the tyrosine kinase by epidermal growth factor was indistinguishable from the second-order rate constant for the dimerization induced by epidermal growth factor. Therefore, dimerization of epidermal growth factor receptor and activation of its tyrosine kinase are coincident events, both initiated by the binding of epidermal growth factor.     

Hydrolysis of p-NN''-phenylenebismaleimide and its adducts with cysteine. Implications for cross-linking of proteins.

To understand the extent of the cross-linking of proteins by the bifunctional reagent p-NN'-phenylenebismaleimide, a quantitative study of competing reactions has been undertaken. The two reactive maleimide rings of the bismaleimide are hydrolysed in mildly alkaline aqueous solutions much more rapidly than is the single maleimide ring of the monofunctional analogue N-ethylmaleimide. The kinetics of hydrolysis are second-order, depending on both imide and hydroxyl ion concentration in the pH range 8-10. The hydrolysis of the first imide ring of the bismaleimide is more rapid than the second, with second-order rate constants of 1600 M-1 . s-1 and 500 M-1 . s-1 respectively, at 25 degrees C. The half-times for hydrolysis of the first and second imide rings at pH 9.0 are therefore only 43s and 140s. Because it renders the maleimide ring unreactive towards cysteine, this rapid hydrolysis can limit the extent of cross-linking of proteins by the bismaleimide.     

Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin

Accurate and precise determinations of thermodynamic parameters of binding are important steps toward understanding many biological mechanisms. Here, a multi-method approach to binding analysis is applied and a detailed error analysis is introduced. Using this approach, the binding thermodynamics and kinetics of the reconstitution of the protein monellin have been quantitatively determined in detail by simultaneous analysis of data collected with fluorescence spectroscopy, surface plasmon resonance and isothermal titration calorimetry at 25 degrees C, pH 7.0 and 150 mM NaCl. Monellin is an intensely sweet protein composed of two peptide chains that form a single globular domain. The kinetics of the reconstitution reaction are slow, with an association rate constant, k(on) of 8.8 x 10(3) M(-1) s(-1) and a dissociation rate constant, k(off) of 3.1 x 10(-4) s(-1). The equilibrium constant K(A) is 2.8 x 10(7) M(-1) corresponding to a standard free energy of association, DeltaG degrees , of -42.5 kJ/mol. The enthalpic component, DeltaH degrees , is -18.7 kJ/mol and the entropic contribution, DeltaS degrees , is 79.8 J mol(-1) K(-1) (-TDeltaS degrees = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropic factors.     

Biogenesis of iron-sulfur clusters in photosystem I: holo-NfuA from the cyanobacterium Synechococcus sp. PCC 7002 rapidly and efficiently transfers [4Fe-4S] clusters to apo-PsaC in vitro

The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. To determine the properties of NfuA, recombinant NfuA from Synechococcus sp. PCC 7002 was overproduced and purified. In vitro reconstituted NfuA contained oxygen- and EDTA-labile Fe/S cluster(s), which had EPR properties consistent with [4Fe-4S] clusters. After reconstitution with 57Fe2+, M?ssbauer studies of NfuA showed a broad quadrupole doublet that confirmed the presence of [4Fe-4S]2+ clusters. Native gel electrophoresis under anoxic conditions and chemical cross-linking showed that holo-NfuA forms dimers and tetramers harboring Fe/S cluster(s). Combined with iron and sulfide analyses, the results indicated that one [4Fe-4S] cluster was bound per NfuA dimer. Fe/S cluster transfer from holo-NfuA to apo-PsaC of PS I was studied by reconstitution of PS I complexes using P700-F(X) core complexes, PsaD, apo-PsaC, and holo-NfuA. Electron transfer measurements by time-resolved optical spectroscopy showed that holo-NfuA rapidly and efficiently transferred [4Fe-4S] clusters to PsaC in a reaction that required contact between the two proteins. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [F(A)/F(B)](-) to P700+ and light-induced low-temperature EPR spectra. These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro.     

Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation

FXYD10 is a 74 amino acid small protein which regulates the activity of shark Na,K-ATPase. The lipid dependence of this regulatory interaction of FXYD10 with shark Na,K-ATPase was investigated using reconstitution into DOPC/cholesterol liposomes with or without the replacement of 20 mol % DOPC with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino acids of FXYD10 had been cleaved by mild, controlled trypsin treatment. Several kinetic properties of the Na,K-ATPase reaction cycle as well as the FXYD-regulation of Na,K-ATPase activity were found to be affected by acidic phospholipids like PI, PS, and PG. This takes into consideration the Na+ and K+ activation, the K+-deocclusion reaction, and the poise of the E1/E2 conformational equilibrium, whereas the ATP activation was unchanged. Anionic phospholipids increased the intermolecular cross-linking between the FXYD10 C-terminus (Cys74) and the Cys254 in the Na,K-ATPase A-domain. However, neither in the presence nor in the absence of anionic phospholipids did protein kinase phosphorylation of native FXYD10, which relieves the inhibition, affect such cross-linking. Together, this seems to indicate that phosphorylation involves only modest structural rearrangements between the cytoplasmic domain of FXYD10 and the Na,K-ATPase A-domain.     

Cytochrome c and c2 binding dynamics and electron transfer with photosynthetic reaction center protein and other integral membrane redox proteins

To further the understanding of the details of c-type cytochrome action as a redox carrier between major electron-transfer proteins, the single-turnover kinetics time course of cytochrome c and cytochrome c2 oxidation by flash-activated photosynthetic reaction center (purified from the bacterium Rhodobacter sphaeroides) has been examined under a wide variety of conditions of concentration, ionic strength, and viscosity with reaction center present in detergent dispersion and phosphatidylcholine proteoliposomes. We find that the three-state model proposed by Overfield and Wraight [Overfield, R. E., & Wraight, C. A. (1980) Biochemistry 19, 3322-3327] is generally sufficient to model the kinetics time course; many similarities are found with the cytochrome c-cytochrome c oxidase interaction in mitochondria. Further, we find the following: (1) Significant "product inhibition" by oxidized cytochrome c (c2) bound to the reaction center is apparent. (2) The viscosity sensitivity of the electron transfer into the reaction center from bound cytochrome c (c2) suggests a physical interpretation of the distal state. (3) The exchange dynamics of oxidized and reduced cytochrome c (c2) are similar regardless of the state of activation of the reaction center. (4) Preferential binding of the oxidized form of cytochrome c is revealed upon reconstitution of the reaction center into neutral lipid vesicles, permitting an independent confirmation of the binding suggested by the kinetics. (5) Flash-activated electron-transfer kinetics in reaction center hybrid protein systems have shown that diffusion and competitive binding characterize the behavior of cytochrome c as a redox carrier between the reaction center protein and either the cytochrome bc1 complex or the cytochrome c oxidase.     

The mechanism of amyloid-fibril formation by stefin B: temperature and protein concentration dependence of the rates

Cystatins, a family of structurally related cysteine proteinase inhibitors, have proved to be useful model system to study amyloidogenesis. We have extended previous studies of the kinetics of amyloid-fibril formation by human stefin B (cystatin B) and some of its mutants, and proposed an improved model for the reaction. Overall, the observed kinetics follow the nucleation and growth behavior observed for many other amyloidogenic proteins. The minimal kinetic scheme that best fits measurements of changes in CD and thioflavin T fluorescence as a function of protein concentration and temperature includes nucleation (modeled as N(I) irreversible transitions with equivalent rates (k(I)), which fitted with N(I) = 64), fibril growth and nonproductive oligomerization, best explained by an off-pathway state with a rate-limiting escape rate. Three energies of activation were derived from global fitting to the minimal kinetic scheme, and independently through the fitting of the individual component rates. Nucleation was found to be a first-order process within an oligomeric species with an enthalpy of activation of 55 +/- 4 kcal mol(-1). Fibril growth was a second-order process with an enthalpy of activation (27 +/- 5 kcal mol(-1)), which is indistinguishable from that of tetramer formation by cystatins, which involves limited conformational changes including proline trans to cis isomerization. The highest enthalpy of activation (95 +/- 5 kcal mol(-1) at 35 degrees C), characteristic of a substantial degree of unfolding as observed prior to domain-swapping reactions, equated with the escape rate of the off-pathway oligomeric state.     

Electrostatic forces contribute to interactions between trp repressor dimers.

The trp repressor of Escherichia coli (TR), although generally considered to be dimeric, has been shown by fluorescence anisotropy of extrinsically labeled protein to undergo oligomerization in solution at protein concentrations in the micromolar range (Fernando, T., and C. A. Royer 1992. Biochemistry. 31:3429-3441). Providing evidence that oligomerization is an intrinsic property of TR, the present studies using chemical cross-linking, analytical ultracentrifugation, and molecular sieve chromatography demonstrate that unmodified TR dimers form higher order aggregates. Tetramers and higher order species were observed in chemical cross-linking experiments at concentrations between 1 and 40 microM. Results from analytical ultracentrifugation and gel filtration chromatography were consistent with average molecular weight values between tetramer and dimer, although no plateaus in the association were evident over the concentration ranges studied, indicating that higher order species are populated. Analytical ultracentrifugation data in presence of corepressor imply that corepressor binding destabilizes the higher order aggregates, an observation that is consistent with the earlier fluorescence work. Through the investigation of the salt and pH dependence of oligomerization, the present studies have revealed an electrostatic component to the interactions between TR dimers.     

High-specific-activity probes and a high-resolution in-gel photo cross-linking assay for protein-DNA complexes

Photochemical cross-linking has been widely employed to identify proteins interacting with specific sites on DNA. Identification of bound proteins usually relies on transfer of a radiolabel from the DNA to the protein by cross-linking. We set out to fine-map a small viral replication preinitiation complex composed of two protein dimers bound to DNA, the bovine papillomavirus E1E2-ori complex. Here we describe a simple method for generating high-specific-activity probes with a phenyl-azide photoactivatible cross-linking group positioned immediately adjacent to a labeled nucleotide. The method is based on the selective destruction of one 5'-phosphorylated strand of a polymerase chain reaction product with lambda exonuclease and reconstitution of the probe with a phosphorothioate-substituted oligonucleotide, an [alpha-(32)P]dNTP, and thermophilic enzymes. We also developed a high-resolution in-gel cross-linking assay to probe defined protein-DNA complexes. With these methods we have obtained structural information for the papillomavirus E1E2-ori preinitiation complex that would otherwise have been hard to obtain. These approaches should be widely applicable to the study of protein-DNA complexes.     

Reduction and oxidation of the active site iron in tyrosine hydroxylase: kinetics and specificity

Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze-quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 +/- 0.1 mM(-)(1) s(-)(1). 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 +/- 0.1 mM(-)(1) s(-)(1) and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated K(m) for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH(4) does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM(-)(1) s(-)(1). S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.     

Reactions of yeast thioredoxin peroxidases I and II with hydrogen peroxide and peroxynitrite: rate constants by competitive kinetics

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. Likely to be critical for both functions is a rapid reaction with hydrogen peroxide, typically with second-order rate constants higher than 10(5) M(-1) s(-1). Until recently, however, the values reported for these rate constants have been in the range of 10(4)-10(5) M(-1) s(-1), including those for cytosolic thioredoxin peroxidases I (Tsa1) and II (Tsa2) from Saccharomyces cerevisiae. To resolve this apparent paradox, we developed a competitive kinetic approach with horseradish peroxidase to determine the second-order rate constant of the reaction of peroxiredoxins with peroxynitrite and hydrogen peroxide. This method was validated and allowed for the determination of the second-order rate constant of the reaction of Tsa1 and Tsa2 with peroxynitrite (k approximately 10(5) M(-1) s(-1)) and hydrogen peroxide (k approximately 10(7) M(-1) s(-1)) at pH 7.4, 25 degrees C. It also permitted the determination of the pKa of the peroxidatic cysteine of Tsa1 and Tsa2 (Cys47) as 5.4 and 6.3, respectively. In addition to providing a useful method for studying thiol protein kinetics, our studies add to recent reports challenging the popular belief that peroxiredoxins are poor enzymes toward hydrogen peroxide, as compared with heme and selenium proteins.     

The role of nanometer-scaled ligand patterns in polyvalent binding by large mannan-binding lectin oligomers

Mannan-binding lectin (MBL) is an important protein of the innate immune system and protects the body against infection through opsonization and activation of the complement system on surfaces with an appropriate presentation of carbohydrate ligands. The quaternary structure of human MBL is built from oligomerization of structural units into polydisperse complexes typically with three to eight structural units, each containing three lectin domains. Insight into the connection between the structure and ligand-binding properties of these oligomers has been lacking. In this article, we present an analysis of the binding to neoglycoprotein-coated surfaces by size-fractionated human MBL oligomers studied with small-angle x-ray scattering and surface plasmon resonance spectroscopy. The MBL oligomers bound to these surfaces mainly in two modes, with dissociation constants in the micro to nanomolar order. The binding kinetics were markedly influenced by both the density of ligands and the number of ligand-binding domains in the oligomers. These findings demonstrated that the MBL-binding kinetics are critically dependent on structural characteristics on the nanometer scale, both with regard to the dimensions of the oligomer, as well as the ligand presentation on surfaces. Therefore, our work suggested that the surface binding of MBL involves recognition of patterns with dimensions on the order of 10-20 nm. The recent understanding that the surfaces of many microbes are organized with structural features on the nanometer scale suggests that these properties of MBL ligand recognition potentially constitute an important part of the pattern-recognition ability of these polyvalent oligomers.     

Fluorescence polarization and intensity kinetic studies of antifluorescein antibody obtained at different stages of the immune response.

Kinetic studies of reactions between fluorescein and antifluorescein antibody produced during early, intermediate, and late stages of the immune response have been carried out utilizing both fluorescence intensity and polarization measurements in the static (time constant similar to 5 sec) and in the stopped-flow modes (time constant similar to 5 msec). During maturation of the immune response, it was found that the "on" second-order association rate constant increased its value only by a factor of three, whereas the "off" dissociation first-order rate constant decreased by a factor of over 1000. Hence, it is the rate of dissociation which largely determines the stability of the hapten-antihapten complex. Furthermore, since second-order rate behavior was found for even heterogeneous antibody, most of the heterogeneity with respect to binding affinity occurs as a result of the heterogeneity in the rate of dissociation of the hapten-antihapten complex and not from the primary combination of hapten and antibody. Antifluorescein antibody which exhibits both high binding affinity (K similar to 5 x 10(11) M-1) and homogeneity with respect to equilibrium binding has been shown to obey second-order association kinetics over wide ranges in concentration. Despite the fact that the value of the second-order rate constant for this fluorescein-antifluorescein reaction is as large as that for most other hapten-antihapten reactions (1.4 x 10(8) M-1 sec-1), the binding reaction has an appreciable activation energy (7 kcal/mol). This is true for both divalent and univalent antibody. Furthermore, the reaction rate parameters are markedly affected by specific anions. The value of the second-order rate constant (18.5 degrees) increases according to the following scheme: salicylate less than trichloroacetate less than SCN- less than ClO4- less than Cl- less than F- less than phosphate. The activation energy increases as follows: trichloroacetate less than phosphate less than F- less than Cl- less than ClO4- less than SCN- less than salicylate, whereas estimates of the entropy of activation indicate that deltaS++ increases as follows: tricholroacetate less than phosphate similar to F- less than Cl- less than ClO4- less than SCN- less than salicylate. The same mechanism which was previously proposed by us for the antigen-antibody reaction is also consistent with the kinetics of the fluorescein-antifluorescein reaction. This mechanism postulates a bimolecular process with structural rearrangements (conformational changes and/or the loss of water) in the formation of the transition state complex. The reaction between the fluorescein hapten and its antibody hence is not diffusion limited.