Assignment of a component of serine proteinase fluorescence spectrum to a cluster of tryptophan residues

An increasing body of information regarding risk factors for perinatal HIV transmission suggests the use of logical management strategies during the prenatal period and parturition directed at maximizing maternal health and minimizing perinatal transmission. This article reviews the recommendations for pharmacologic therapy and rational obstetric management strategies to decrease perinatal HIV transmission based on published clinical trials and a review of data relevant to transmission.     

The binding of 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate by human antithrombin III

The binding of 1-anilino-8-naphthalenesulfonate to human antithrombin III was studied by fluorescence enhancement of the fluorophor and fluorescence quenching of the protein emission. Two molecules of 1-anilino-8-naphthalenesulfonate were found to bind per antithrombin molecule with an average dissociation constant of 4.4-10(-5) M. The binding of heparin to antithrombin was studied by ultraviolet difference spectroscopy. The stoichiometry of the heparin binding indicated 1.8 binding sites with an average dissociation constant of 4.3 - 10(-6) M. Further the fluorometric competition experiments with 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate indicated two different classes of anion binding sites on the antithrombin molecule.     

Mechanism of heparin activation of antithrombin. Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.     

The N-terminal segment of antithrombin acts as a steric gate for the binding of heparin

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high-affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N-terminal segment of antithrombin, residues 22-46 (segment 22-46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29-30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5-fold lowered affinity for heparin pentasaccharide and 1.8-fold for full-length, high-affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22-46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22-46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N-terminal cleavage site, implying that segment 22-46 moves to interact with heparin in the conformational change and thus stabilizes the complex.     

Oxidation of apolipoprotein(a) inhibits kringle-associated lysine binding: the loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide-linked to apolipoprotein B-100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2'-azobis-(amidinopropane) HCl (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r-apo(a), to bind through their LBS domains. AAPH treatment caused a time-dependent decrease in the number of functional Lp(a) or r-apo(a) molecules capable of binding to fibrin or lysine-Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r-apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle-containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r-apo(a) was observed as assessed by native or SDS-PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl-containing compounds increase the LBS affinity of Lp(a) for fibrin.     

Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase

The atypical variant of human butyrylcholinesterase has Gly in place of Asp 70. Patients with this D70G mutation respond abnormally to the muscle relaxant succinyldicholine, experiencing hours of apnea rather than the intended 3 min. Asp 70 is at the rim of the active site gorge 12 A from the active site Ser 198. An unanswered question in the literature is why the atypical variant has a 10-fold increase in Km for compounds with a single positive charge but a 100-fold increase in Km for compounds with two positive charges. We mutated residues Asp 70, Trp 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in mammalian cells. Steady-state kinetic parameters for hydrolysis of butyrylthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl butyrate were determined. The wild type and the D70G mutant had identical k(cat) values for all substrates. Molecular modeling and molecular dynamics suggested that succinyldicholine could bind in two consecutive orientations in the active site gorge; formation of one complex caused a conformational change in the omega loop involving Asp 70 and Trp 82. We propose the formation of three enzyme-substrate intermediates preceding the acyl-enzyme intermediate; kinetic data support this contention. Substrates with a single positive charge interact with Asp 70 just once, whereas substrates with two positive charges, for example succinyldithiocholine, interact with Asp 70 in two complexes, thus explaining the 10- and 100-fold increases in Km in the D70G mutant.     

Site-directed mutagenesis of recombinant human beta 2-glycoprotein I identifies a cluster of lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity

beta2-Glycoprotein I (beta2GPI) is a phospholipid-binding serum protein with anticoagulant properties. It plays a vital role in the binding of anti-cardiolipin Abs purified from patients with autoimmune disease when assayed in a cardiolipin (CL) ELISA. Based on a three-dimensional model of beta2GPI, electrostatic calculations, and earlier peptide studies, a highly positively charged amino acid sequence, Lys282-Asn-Lys-Glu-Lys-Lys287, located in the fifth domain of beta2GPI, has been predicted to be the phospholipid binding site. We tested this hypothesis by site-directed mutagenesis of residues in the predicted phospholipid binding site and by assessing the mutants for phospholipid binding and anti-beta2GPI activity. A single amino acid change from Lys286 to Glu significantly decreased the binding of beta2GPI to CL. Double and triple mutants 2k (from Lys286, 287 to Glu286, 287), 2ka (from Lys284, 287 to Glu284, 287), and 3k (from Lys284, 286, 287 to Glu284, 286, 287) possessed no binding of Ab to beta2GPI in a CL ELISA, as well as no inhibitory activity on the binding of iodinated native beta2GPI to CL. These results indicate that the residues Lys284, Lys286, and Lys287 in the fifth domain of beta2GPI are critical for its binding to anionic phospholipids and its subsequent capture for binding of anti-beta2GPI Abs.     

Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli

Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx. In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes. Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax. Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substrate protection against [14C]-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca. 80 microM). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity.     

Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle

Isolates of foot-and-mouth disease virus (FMDV) exist as complex mixtures of variants. Two different serotype O1 Campos preparations that we examined contained two variants with distinct plaque morphologies on BHK cells: a small, clear-plaque virus that replicates in BHK and CHO cells, and a large, turbid-plaque virus that only grows in BHK cells. cDNAs encoding the capsids of these two variants were inserted into a genome-length FMDV type A12 infectious cDNA and used to produce chimeric viruses that exhibited the phenotype of the original variants. Analyses of these viruses, and hybrids created by exchanging portions of the capsid gene, identified codon 56 in VP3 (3056) as the critical determinant of both cell tropism and plaque phenotype. Specifically, the CHO growth/clear-plaque phenotype is dependent on the presence of the highly charged Arg residue at 3056, and viruses with this phenotype and genotype were selected during propagation in tissue culture. The genetically engineered Arg 3056 virus was highly attenuated in bovines, but viruses recovered from animals inoculated with high doses of this virus had lost the ability to grow in CHO cells and contained either an uncharged residue at 3056 or a negatively charged Glu substituted for a Lys at a spatially and antigenically related position on VP2 (2134). Comparison of these animal-derived viruses to other natural and engineered viruses demonstrated that positively charged residues are required at both 2134 and 3056 for binding to heparin. Taken together, these results indicate that in vitro cultivation of FMDV type O selects viruses that bind to heparin and that viruses with the heparin-binding phenotype are attenuated in the natural host.     

Mutagenesis and modeling of the neurotensin receptor NTR1. Identification of residues that are critical for binding SR 48692, a nonpeptide neurotensin antagonist

The two neurotensin receptor subtypes known to date, NTR1 and NTR2, belong to the family of G-protein-coupled receptors with seven putative transmembrane domains (TM). SR 48692, a nonpeptide neurotensin antagonist, is selective for the NTR1. In the present study we attempted, through mutagenesis and computer-assisted modeling, to identify residues in the rat NTR1 that are involved in antagonist binding and to provide a tentative molecular model of the SR 48692 binding site. The seven putative TMs of the NTR1 were defined by sequence comparison and alignment of bovine rhodopsin and G-protein-coupled receptors. Thirty-five amino acid residues within or flanking the TMs were mutated to alanine. Additional mutations were performed for basic residues. The wild type and mutant receptors were expressed in COS M6 cells and tested for their ability to bind 125I-NT and [3H]SR 48692. A tridimensional model of the SR 48692 binding site was constructed using frog rhodopsin as a template. SR 48692 was docked into the receptor, taking into account the mutagenesis data for orienting the antagonist. The model shows that the antagonist binding pocket lies near the extracellular side of the transmembrane helices within the first two helical turns. The data identify one residue in TM 4, three in TM 6, and four in TM 7 that are involved in SR 48692 binding. Two of these residues, Arg327 in TM 6 and Tyr351 in TM 7, play a key role in antagonist/receptor interactions. The former appears to form an ionic link with the carboxylic group of SR 48692, as further supported by structure-activity studies using SR 48692 analogs. The data also show that the agonist and antagonist binding sites in the rNTR1 are different and help formulate hypotheses as to the structural basis for the selectivity of SR 48692 toward the NTR1 and NTR2.     

不锈钢的X-射线荧光光谱分析及其牌号的自动模糊识别研究

在建立了X-射线荧光光谱分析不锈钢中Cr,Ni,Mn,Mo,Cu,Co,Nb,Ti,V,W,Si,Al,P,S,Sn,As,Fe共17个元素含量方法的基础上,创建了能自动模糊识别不锈钢牌号的分析检索程序,使X-射线荧光光谱仪在完成不锈钢样品测量后,能自动显示出与所检样品相符的钢号及其标准化学成分数据。     

Identification of equivalent residues in the gamma, delta, and epsilon subunits of the nicotinic receptor that contribute to alpha-bungarotoxin binding

Cysteine was introduced from residues 116 to 121 of the gamma subunit of the fetal mouse acetylcholine receptor, and the mutant receptors were treated with methanethiosulfonate reagents and examined for changes in ligand binding properties. Of the 18 combinations of mutant and reagent, only receptors harboring gammaL119C treated with the quaternary ammonium reagent MTSET (trimethylammonium-ethyl methanethiosulfonate) show a decreased number of alpha-bungarotoxin (alpha-btx) sites. The decrease of 50% suggests that alpha-btx binding to the site harboring gammaL119C is blocked. Analysis of binding of the site-selective ligands dimethyl-d-tubocurarine (DMT) and alpha-conotoxin M1 (CTX) confirm specificity of modification for the site harboring gammaL119C. Cysteines placed at equivalent positions of the delta and epsilon subunits also lead to selective loss of alpha-btx binding following MTSET treatment. gammaL119C receptors treated with the primary amine reagent MTSEA (aminoethyl methanethiosulfonate) retain alpha-btx binding to both sites but show reduced affinity for DMT and CTX at the modified site. Lysine mutagenesis of Leugamma119, Leudelta121, and Leuepsilon119 mimics MTSEA treatment, whereas mutagenesis of Thralpha119 and Glnbeta119 is without effect, demonstrating subunit and residue specificity of MTSEA modification. MTSET modification of nearby gammaY117C does not block alpha-btx binding but markedly diminishes affinity for DMT and CTX. The overall findings indicate a localized point of interaction between alpha-btx and the modified gammaL119C, deltaL121C, and epsilonL119C.     

Tyrosine sulfation of the glycoprotein Ib-IX complex: identification of sulfated residues and effect on ligand binding

Here, we present evidence that glycoprotein (GP) Ib alpha, one of three polypeptides that make up the GP Ib--IX complex--the receptor for von Willebrand factor (vWf) on the surface of unactivated platelets--is modified by sulfation of tyrosine residues. Only GP Ib alpha was found to incorporate 35S when the GP Ib--IX complex was immunoprecipitated from [35S]sulfate metabolically labeled L and CHO cells that express the recombinant complex. The occurrence of sulfation on tyrosine residues of the polypeptide backbone was determined by removing O- and N-linked oligosaccharides. Limited proteolytic digestion of metabolically labeled GP Ib alpha revealed that sulfated tyrosine residues are located in the 45-kDa globular region containing the vWf binding site. By mutating potentially sulfated tyrosine residues to phenylalanine and comparing the stoichiometry of sulfate incorporation of these mutants to the incorporation in wild-type GP Ib alpha, three clustered tyrosine residues--Tyr-276, Tyr-278, and Tyr-279-were identified that undergo the modification. Culturing cells in sulfate-depleted medium containing sodium chlorate and guaiacol completely inhibited GP Ib alpha sulfation but did not decrease GP Ib-IX expression on the cell surface. Similarly, transiently transfected CHO cells expressed the mutant GP Ib alpha polypeptide on their surfaces at the same levels as they expressed wild-type GP Ib alpha. These results suggest that tyrosine sulfation of GP Ib alpha has little or no effect on the synthesis, assembly, and surface expression of the GP Ib-IX complex. Nevertheless, inhibiting sulfation of GP Ib alpha reduced the binding of 125I-labeled vWf in the presence of ristocetin by up to 37%.(ABSTRACT TRUNCATED AT 250 WORDS)     

WIN 35,428 and mazindol are mutually exclusive in binding to the cloned human dopamine transporter

It has been suggested that cocaine and mazindol bind to separate sites on the dopamine transporter. In the present study, we address this issue by examining the inhibition by mazindol of the binding of [3H]WIN 35,428 ([3H]2beta-carbomethyoxy-3beta-(4-fluorophenyl)-tropane), a phenyltropane analog of cocaine, and the inhibition by WIN 35,428 of [3H]mazindol binding to the cloned human dopamine transporter expressed in C6 glioma cells. The design involved the construction of inhibition curves at six widely different radioligand levels, enabling the distinction between the nonlinear hyperbolic competition (i.e., negative allosteric) model and the competitive (i.e., mutually exclusive binding) model. Nonlinear computer curve-fitting analysis indicated no difference in the goodness of fit between the two models; the negative allosteric model indicated an extremely high allosteric constant of approximately > or = 100, which practically equates to the competitive model. The present results suggest that complex interactions reported between cocaine and mazindol in inhibiting dopamine transport are beyond the level of ligand recognition.     

Beta PDGFR-IgG chimera demonstrates that human beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding

To localize human beta PDGFR binding determinants, we constructed a fusion protein comprising beta PDGFR Ig-like domains 1 to 3 and an IgG1 Fc domain (beta PDGFR-HFc). beta PDGFR-HFc was expressed as a 200 kDa dimeric molecule and contained Fc epitopes as demonstrated by anti-mouse Fc antibody recognition. Scatchard analysis revealed that PDGF BB possessed a dissociation constant of 1.5 nM for beta PDGFR-HFc. Thus, beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding. We exploited this fusion protein technology to identify and characterize beta PDGFR antagonists using a sensitive beta PDGFR immunosorbent assay. In this assay, beta PDGFR-HFc half-maximally bound to PDGF BB with an affinity of around 150 pM. Suramin, as well as bacterially expressed and refolded human alpha PDGFR domains 1-3, inhibited beta PDGFR-HFc binding to PDGF BB half-maximally at 25 microM and 10 nM respectively. Therefore, alpha PDGFR D1-3, like beta PDGFR D1-3, are sufficient for high affinity PDGF BB binding. Furthermore, the beta PDGFR-HFc immunosorbent assay will be useful to identify beta PDGFR antagonists as well as to study alpha and beta PDGFR substitution mutants which further map receptor binding determinants.     

Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity

We have identified mutations in Raf-1 that increase binding to Ras. The mutations were identified making use of three mutant forms of Ras that have reduced Raf-1 binding (Winkler, D. G., Johnson, J. C., Cooper, J. A., and Vojtek, A. B. (1997) J. Biol. Chem. 272, 24402-24409). One mutation in Raf-1, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation. Missense substitutions of residues 143 and 144 in the Raf-1 cysteine-rich domain were isolated multiple times. These Raf-1 mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras. Each was slightly activated relative to wild-type Raf-1 in a transformation assay. In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes. Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-1. We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-1 is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-1 and increasing Raf-1 activity.     

Two regions of the Mn-stabilizing protein from Synechococcus elongatus that are involved in binding to photosystem II complexes

Limited proteolysis of the Mn-stabilizing protein (MSP) from the thermophilic cyanobacterium Synechococcus elongatus with chymotrypsin, trypsin or lysylendopeptidase that yielded four major polypeptides of 26 kDa, 22 kDa, 15 kDa and 11 kDa on denaturing gel electrophoresis resulted in total loss of the binding capacity of the protein to PSII complexes. Analyses of electrophoretic patterns and amino-terminal sequences of the proteolytic products revealed that the three proteases specifically cleaved the protein at a site between Phe156 and Gly163 or between Arg184 and Ser191. Site-directed mutagenesis was used to construct two mutant MSPs that had a nick between Phe156 and Leu157, a chymotrypsin-cleavage site, and Met before Leu157 or in place of Leu157. The two mutant proteins failed to bind to PSII complexes, although they largely retained ordered secondary structure and comigrated with the wild-type proteins in non-denaturing gel electrophoresis. The loss of the protein binding can be ascribed to introduction of a nick because a mutant protein that had Met in place of Leu157 but no nick was able to specifically bind to the functional site of PSII complexes and restore the oxygen-evolving activity as effectively as the wild-type protein. In contrast, a mutant MSP with Met inserted between Phe156 and Leu157 bound only weakly and non-specifically to PSII complexes and failed to reactivate oxygen evolution. Thus, the binding of the protein to the functional site of the PSII complex was highly sensitive to a small structural change that was caused by cleavage or insertion of a single amino acid residue between Phe156 and Leu157. The results suggest that the Phe156-Gly163 and Arg184-Ser191 sequences of the cyanobacterial MSP are regions for interaction with PSII complexes.     

Role of polymorphic residues of human leucocyte antigen-DR molecules on the binding of human immunodeficiency virus peptides

Short peptides which contained a single Cys residue were introduced into both N- and C-termini of the Cys-free mutant of DHFR (Cys85 --> Ala, Cys152 --> Ser double mutant) by a recombinant DNA method, then the terminal regions were connected through a disulfide bond by oxidation. The oxidized form and reduced form proteins have as high enzymatic activity as wild-type DHFR. There is no detectable difference between the CD spectra of the reduced and oxidized forms at low (15 degrees C, native condition) and high temperature (80 degrees C, unfolded condition). The thermal transition of the oxidized proteins at the concentration of 0.15 mg/ml (8.5 microM) is completely reversible as demonstrated by the CD spectra. No aggregated materials were detected in the oxidized protein on gel-filtration HPLC after heat treatment up to the protein concentration of 0.5 mg/ml. The reduced protein, however, even in the presence of reducing agent, showed only partial reversibility, with as much as 55 and 95% of the heat-treated protein at the concentrations of 0.15 and 0.5 mg/ml being eluted as the high molecular aggregated form, respectively. The apparent transition temperatures (Tm) of the oxidized forms were 5-7 degrees C higher than those of the reduced counterparts. The oxidized protein that had been denatured with guanidine-HCl was eluted later than the denatured reduced protein on gel-filtration HPLC in the presence of 5 M guanidine-HCl. The limitation of spatial movement of the termini may prevent intermolecular interaction of exposed domains during denaturation-renaturation process, giving rise to the irreversible denaturation. The flexibility of the terminal is also suggested to be an important factor for improving thermal stability of proteins.     

Haemoglobin binding with haptoglobin. Unequivocal demonstration that the beta-chains of human haemoglobin bind to haptoglobin

Haptoglobin binding to haemoglobin and its isolated alpha- and beta-chains was studied by use of a highly sensitive solid-phase radiometric assay. As expected, adsorbents of haemoglobin bound 125I-labelled haptoglobin more efficiently than did adsorbents of the alpha-chain. However, unexpectedly, adsorbents of the beta-chain were found to be essentially identical with those of the alpha-chain in their ability to bind haptoglobin. These results demonstrate, unequivocally, the ability of beta-chains to bind to haptoglobin, and indicate that this assay is particularly convenient and useful for studying haptoglobin interactions with haemoglobin and its alpha- and beta-chains.     

Ultraviolet difference spectroscopy study of alpha- and beta gamma-thrombin binding to heparin

The interaction of alpha- and beta gamma-thrombin with heparin was studied by ultraviolet difference spectroscopy within the wavelength range of 230-300 nm. The absorption difference spectrum of the thrombin-heparin complex was negative and had two maxima at 255 nm (5300 M-1 cm-1) and 282 nm (4700 M-1 cm-1) for alpha-thrombin and at 240 nm (4900 M-1 cm-1) and 282 nm (4100 M-1 cm-1) for beta gamma-thrombin. It is assumed that the conformational changes induced by heparin in the enzyme molecule involve the transfer of some tryptophan and tyrosine residues from the interior of the protein to the surface. The absorption changes during alpha-thrombin--heparin interaction at physiological ionic strength suggest binding of some alpha-thrombin molecules to a heparin molecule at the ligand-enzyme molar ratio lower than 1. Under the same conditions beta gamma-thrombin forms an equimolar complex with heparin with the dissociation constant equal to 7,0.10(-9) M. The ionic strength increase up to 0,217 M NaCl results in some disturbances in beta gamma-thrombin-heparin interaction and prevents the binding of additional alpha-thrombin molecules to an equimolar complex of alpha-thrombin with heparin. Therefore the kinetics of the two enzyme forms interaction with heparin are similar, the alpha-thrombin affinity for heparin being a little higher. The data obtained suggest that alpha-thrombin binding to heparin is essential for biological inactivation of thrombin.