Proximity of thiol esters and bait region in human alpha 2-macroglobulin: paramagnetic mapping

The two key structural features of alpha 2-macroglobulin (alpha 2M) involved in inhibitory caging of proteases are the thiol ester and the bait region. This paper examines the environment of the hydrolyzed thiol ester in methylamine-treated human alpha 2M and the separation between the bait region and the thiol ester and between the four thiol esters in the tetramer to try to further our understanding of how bait region proteolysis triggers thiol ester cleavage. The sulfhydryl groups of Cys-949, formed upon cleavage of the thiol ester by methylamine, were specifically labeled with the nitroxide spin-labels 3-(2-iodoacetamido)-PROXYL (iodo-I) (PROXYL = 2,2,5,5-tetramethylpyrrolidine-1-oxyl), 3-[2-(2-iodoacetamido)acetamido]-PROXYL (iodo-II), and 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (iodo-III). ESR spectra of these alpha 2M derivatives showed that label I is firmly held and label II has limited freedom of rotation consistent with location of the cysteine residue in a narrow cavity. Label III has much greater motional freedom. From the absence of dipole-dipole splittings in the ESR spectra, it is concluded that the four nitroxide groups in the tetramer are more than 20 A apart for both label I and label II. Label I broadens 1H NMR signals from one phenylalanyl, one tyrosyl, and four histidyl residues in the bait region. Separations of 11-17 A are estimated between the nitroxide of label I and these residues. Label II is further away and only broadens resonances from one of the histidines.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of the C949S mutant human alpha(2)-macroglobulin demonstrates the critical role of the internal thiol esters in its proteinase-entrapping structural transformation

A three-dimensional reconstruction of a protein-engineered mutant alpha(2)-macroglobulin (alpha(2)M) in which a serine residue was substituted for the cysteine 949 (C949S), making it unable to form internal thiol ester moieties, was compared with native and methylamine-transformed alpha(2)Ms. The native alpha(2)M structure consists of two oppositely oriented Z-shaped strands. Thiol ester cleavage following an encounter with a proteinase or a nucleophilic attack by methylamine causes a structural transformation in which the strands assume an opposite handedness and a significant portion of the protein density migrates from the distal ends of the molecule toward the center. The C949S mutant showed a protein density distribution very similar to that of transformed alpha(2)M, with a compact central region of protein density connected to two receptor-binding arms on each end of the molecule. Since no particle shapes characteristic of native or half-transformed alpha(2)Ms were seen in electron micrographs and the C949S mutant and alpha(2)M-methylamine structures are highly similar, we conclude that the intact thiol esters maintain native alpha(2)M in a quasi-stable state. In their absence, alpha(2)M folds into the more stable transformed structure, which displays the functionally important receptor-binding domains and contains the proteinase-entrapping internal cavity.     

Identification of a monoclonal antibody specific for a neoantigenic determinant on alpha 2-macroglobulin: use for the purification and characterization of binary proteinase-inhibitor complexes

A monoclonal antibody was obtained from the fusion of spleen cells of mice, immunized with methylamine-treated alpha 2-macroglobulin (alpha 2M), with the myeloma cell line P3-X63-Ag8.653. A competitive binding assay demonstrated that the antibody was specific for a neoantigen expressed on alpha 2M when the inhibitor reacts with proteinases or with methylamine. When immobilized, the monoclonal antibody retained its ability to specifically bind alpha 2M-proteinase complexes or methylamine-treated alpha 2M, both of which could be quantitatively recovered from the immunoaffinity column by lowering the pH to 5.0. Binary alpha 2M-proteinase complexes of trypsin, plasmin, and thrombin, prepared by incubating large amounts of alpha 2M with a small amount of enzyme, were isolated by immunoaffinity chromatography. Each purified complex was characterized with regard to proteinase content, extent of alpha 2M subunit cleavage, extent of thiol ester hydrolysis, and extent of conformational change. Each complex contained 0.8-0.9 mol of proteinase/mol of inhibitor. In the alpha 2M-thrombin, alpha 2M-plasmin, and alpha 2M-trypsin complexes, approximately 50%, 60%, and 75% of the subunits are cleaved, respectively. Titration of sulfhydryl groups revealed that all purified binary complexes contained 2 +/- 0.5 mol of thiol/mol of complex, suggesting that each complex retains two intact thiol ester bonds. When the purified complexes were incubated with excess trypsin or with methylamine, an additional 1-2 mol of sulfhydryl/mol of complex could be titrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent probes as a measure of conformational alterations induced by nucleophilic modification and proteolysis of bovine alpha 2-macroglobulin

Conformational alterations occurring in bovine alpha 2-macroglobulin (alpha 2M) resulting from proteolysis and nucleophilic modification have been monitored by UV difference spectra, circular dichroism, and changes in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate (TNS) and bis(8-anilino-1-naphthalenesulfonate) (Bis-ANS). The results of this study indicate that these two dyes appear capable of differentiating between conformational changes induced by proteolysis and those induced by methylamine treatment. It appears that TNS is a sensitive probe for monitoring protease-induced but not methylamine-induced conformational changes in bovine alpha 2M. Bis-ANS, on the other hand, appears suitable for monitoring conformational changes induced by methylamine treatment or proteolysis of the molecule and was used as a probe to monitor the kinetics of the conformational change induced by methylamine treatment. It was found that the conformational change did not occur simultaneously with cleavage of the thiol ester bonds by the nucleophile, measured by titration of free sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate). The data are consistent with a model in which initial nucleophilic attack results in exposure of sulfhydryl groups, resulting in a conformational change measured by an increase in fluorescence. This event is followed by a unimolecular step representing a conformational change in the protein that results in a further increase in the fluorescence signal. The second-order rate constant for hydrolysis of the thiol ester bonds was determined to be 3.4 +/- 1.0 M-1 s-1, while the rate constant for the conformational change was (4.4 +/- 0.8) X 10(-4) s-1.     

Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation

The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Separation and localization of the four cysteine-949 residues in human alpha 2-macroglobulin using fluorescence energy transfer.

By use of the intermediate form (I-form) [Gettins, Crews, & Cunningham (1989) Biochemistry 28, 5613-5618], alpha 2-macroglobulin can be specifically labeled with fluorescent probes in a manner that allows the determination of the topology of the four thiol ester derived Cys949 residues within this large tetrameric protease inhibitor. Freshly prepared I-form alpha 2-macroglobulin was reacted with 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonate (1,5-I-AEDANS) to produce alpha 2-macroglobulin specifically and stoichiometrically labeled with 1,5-AEDANS (donor) at the two Cys949 SH groups in the first protease interaction site. Upon subsequent reaction of this labeled species with chymotrypsin, the remaining two bait regions and thiol ester linkages were opened, generating two free SH groups on the two Cys949 residues in the second protease interaction site. These SH groups were specifically and stiochiometrically labeled with 5-(iodoacetamido)fluorescein (acceptor). Fluorescence energy transfer from donor to acceptor results in 82% loss of AEDANS fluorescence intensity. By use of an R0(2/3) value of 43.5 A, calculated from the spectral parameters of this system, an R(2/3) separation between donor and acceptor of 33.9 A was calculated. From fluorescence anisotropy measurements of both donor and acceptor attached to alpha 2-macroglobulin, upper and lower limits on the separation of 43.4 and 26.1 A, respectively, were calculated. These separations, small in the context of the alpha 2-macroglobulin tetramer, which has approximate dimensions of 190 x 90 x 90 A, severely restrict the possible locations of the four Cys949 residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in the proteinase inhibition mechanism of human alpha 2-macroglobulin and pregnancy zone protein.

Different conformational states of human alpha 2-macroglobulin (alpha 2M) and pregnancy zone protein (PZP) were investigated following modifications of the functional sites, i.e. the 'bait' regions and the thiol esters, by use of chymotrypsin, methylamine and dinitrophenylthiocyanate. Gel electrophoresis, mAb (7H11D6 and alpha 1:1) and in vivo plasma clearance were used to describe different molecular states in the proteinase inhibitors. In alpha 2M, in which the thiol ester is broken by binding of methylamine and the 'trap' is closed, cyanylation of the liberated thiol group from the thiol ester modulates reopening of the 'trap' and the 'bait' regions become available for cleavage again. The trapping of proteinases in the cyanylated derivative indicates that the trap functions as in native alpha 2M. In contrast, cyanylation has no effect on proteinase-treated alpha 2M. As demonstrated by binding to mAb, the methylamine and dinitrophenylthiocyanate-treated alpha 2M exposes the receptor-recognition site, but the derivative is not cleared from the circulation in mice. The trap is not functional in PZP. In native PZP and PZP treated with methylamine, the conformational states seem similar. The receptor-recognition sites are not exposed and removal from the circulation in vivo is not seen for these as for the PZP-chymotrypsin complex. Tetramers are only formed when proteinases can be covalently bound to the PZP. Conformational changes are not detected in PZP derivatives in which the thiol ester is treated with methylamine and dinitrophenylthiocyanate. The results suggest that the conformational changes in alpha 2M are generated by mechanisms different to these in PZP. The key structure gearing the conformational changes in alpha 2M is the thiol ester, by which the events 'trapping' and exposure of the receptor-recognition site can be separated. In PZP, the crucial step for the conformational changes is the cleavage of the 'bait' region, since cleavage of the thiol ester does not lead to any detectable conformational changes by the methods used.     

Amino acid sequence around the thiolester of alpha 2-macroglobulin from plasma of the crayfish, Pacifastacus leniusculus

Alpha 2-Macroglobulin (alpha 2 M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion-exchange chromatography and gel filtration techniques. The Pacifastacus alpha 2 M molecule (P alpha 2 M) was radio-actively labeled in the thiol ester structure with iodo [14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C-labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human alpha 2 M and related proteins. The N-terminal sequence of P alpha 2 M was related to that recently determined for lobster alpha 2 M [(1987) J. Biol. Chem. 262, 14606-14611].     

Intersubunit cross-linking by cis-dichlorodiammineplatinum(II) stabilizes an alpha 2-macroglobulin "nascent" state: evidence that thiol ester bond cleavage correlates with receptor recognition site exposure

Treatment of human alpha 2-macroglobulin (alpha 2M) with proteinase results in cleavage of the alpha 2M subunits and subsequently in a conformational change in the inhibitor. This change irreversibly traps the proteinase and is accompanied by the generation of four thiol groups as well as exposure of receptor recognition sites. cis-Dichlorodiammineplatinum(II) (cis-DDP) causes extensive intersubunit cross-linking of alpha 2M. Incubation of alpha 2M or cis-DDP-treated alpha 2M with trypsin results in complete subunit cleavage; however, trypsin treatment of cis-DDP-alpha 2M does not result in a conformational change as determined by nondenaturing polyacrylamide gel electrophoresis (PAGE), receptor recognition site exposure, or appearance of thiol groups from the inhibitor. These results are in marked contrast to previous studies which demonstrated that incubation of cis-DDP-treated alpha 2M with CH3NH2 resulted in thiol ester bond cleavage and receptor recognition site exposure. cis-DDP-treated alpha 2M bound only 0.13 mol of 125I-trypsin/mol of cis-DDP-alpha 2M. Incubation of trypsin-treated cis-DDP-alpha 2M with diethyldithiocarbamate (DDC), a potent chelator of platinum compounds, results in the removal of the intersubunit cross-links and completion of the alpha 2M conformational change as determined by nondenaturing PAGE. Complete receptor recognition site exposure and the appearance of 3.3 thiol groups/mol of alpha 2M also occur following this treatment. These results demonstrate that cross-linking of alpha 2M by cis-DDP prevents a conformational change in the inhibitor which is necessary for thiol ester bond activation and cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence similarity between alpha 2-macroglobulin from the horseshoe crab, Limulus polyphemus, and proteins of the alpha 2-macroglobulin family from mammals.

1. Purified alpha 2-macroglobulin (alpha 2M) from the American horseshoe crab, Limulus polyphemus was cleaved with trypsin and 20 of the tryptic peptides were sequenced and compared with the sequences of human alpha 2M, rat alpha 1M, alpha 2M, and alpha 1-inhibitor 3, and human complement proteins C3 and C4. 2. Ten of the peptides (233 residues), including that containing the thiol ester site, could be aligned unambiguously with stretches in mammalian alpha 2M, with a degree of identity greater than 30%. 3. The 12-residue thiol ester-containing peptide of Limulus alpha 2M showed 67% identity with the same stretch of human alpha 2M.     

Interaction of mannan binding lectin with alpha2 macroglobulin via exposed oligomannose glycans: a conserved feature of the thiol ester protein family?

The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor alpha(2) macroglobulin (alpha2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. alpha2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between alpha2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man(5-7) occupying Asn-846 on alpha2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active alpha2M (thiol ester intact) and protease-cleaved alpha2M (thiol ester cleaved). We demonstrate that MBL is able to interact with alpha2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to alpha2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral alpha2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.     

Human alpha2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.     

Spin-label and fluorescence labeling studies of the thioester bonds in human alpha 2-macroglobulin

Upon cleavage of the reactive thioester bonds (Cys-949-Glx-952) of tetrameric human alpha 2-macroglobulin (alpha 2M) by methylamine, one sulfhydryl group per alpha 2M subunit is exposed. These identical sulfhydryl group sites were labeled with the thiol-specific nitroxide spin-labels (1-oxy-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate and (1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)methyl methanethiosulfonate, a homologous series of maleimide spin-labels, and the thiol-specific fluorescent probe 2-[(4-maleimidophenyl)amino]naphthalene-6-sulfonic acid sodium salt (MANS). The ESR and fluorescence results showed that these sulfhydryl group sites were at the base of a narrow crevice that is greater than or equal to 8 A deep. Although the bound MANS fluorophore was slightly blue shifted with an enhanced quantum yield vs the free label in water, the environment of the sulfhydryl site appeared to be of a polar nature when compared with the emission maxima in several solvents of varying polarity. The Glx residue participating in the thioester linkage in the intact protein was labeled with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl. The distance between the Glx and Cys moieties was estimated at greater than or equal to 10-25 A from double spin-labeling experiments.     

Electron microscopical localization of the alpha 2-macroglobulin thiol ester sites

The active thiol ester groups of alpha 2-macroglobulin (alpha 2M) were reacted with a biotin derivative and the sites labelled with avidin-ferritin complexes. Electron micrographs show a strong preference of attachment of the ferritins to the ends of the rods of the H-shaped molecules. A mutual "cross-labelling" was observed in an alpha 2M preparation which yielded dimers of the molecules which must have been formed during purification. The molecules were mostly attached to each other at the ends of the rods of the H-shaped molecules. It is concluded that the thiol esters responsible for the covalent attachment of the proteinases (and other molecules) may be located more in the distal parts of the alpha 2M molecules, while the proteinase molecules are finally trapped near to the centre of the alpha 2M molecules.     

Electron microscopy of alpha 2-macroglobulin with a thiol ester bound ligand.

In order to covalently bind the hydrolyzed thiol ester groups of the human alpha 2-macroglobulin (alpha 2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M(r) = 13,000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed alpha 2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of alpha 2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in alpha 2M. They also suggest that the four thiol esters are located near the center of the alpha 2M molecule.     

Thrombin-induced conformational changes of human alpha 2-macroglobulin: evidence for two functional domains

The interaction of thrombin with alpha 2-macroglobulin (alpha 2M) was characterized by monitoring conformational changes and measuring the increase of free sulfhydryl groups during the reaction. Under experimental conditions where [thrombin] greater than [alpha 2M], the conformational change, measured by increases in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate, and thiol group appearance displayed biphasic kinetics. The initial rapid phase results in the formation of a stable complex, the appearance of two sulfhydryl groups, the cleavage of approximately half of the Mr 180 000 subunits, and a conformational change that is not as extensive as that which occurs with trypsin. The slower phase is associated with the appearance of two additional sulfhydryl groups, increased cleavage of the Mr 180 000 subunit, and additional conformational changes. The available evidence suggests that the slow phase results from hydrolysis of the Mr 180 000 subunit(s) due to proteolysis of the alpha 2M-thrombin complex by free thrombin. Experiments with 125I-thrombin document the binding of 1 mol of thrombin/mol of alpha 2M that is not dissociated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex. At higher ratios of thrombin to alpha 2M, a second mole of thrombin will reversibly associate with the 1:1 alpha 2M-thrombin complex. Under conditions where [thrombin] less than [alpha 2M], biphasic kinetics were not observed, and the conformational change, sulfhydryl appearance, and hydrolysis of the Mr 180 000 subunit were found to follow second-order kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of human blood coagulation factor Xa by alpha 2-macroglobulin

The inactivation of activated factor X (factor Xa) by alpha 2-macroglobulin (alpha 2M) was studied. The second-order rate constant for the reaction was 1.4 X 10(3) M-1 s-1. The binding ratio was found to be 2 mol of factor Xa/mol of alpha 2M. Interaction of factor Xa with alpha 2M resulted in the appearance of four thiol groups per molecule of alpha 2M. The apparent second-order rate constants for the appearance of thiol groups were dependent on the factor Xa concentration. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and factor Xa. Under nonreducing conditions, four factor Xa-alpha 2M complexes were observed. Reduction of these complexes showed the formation of two new bands. One complex (Mr 225,000) consisted of the heavy chain of the factor Xa molecule covalently bound to a subunit of alpha 2M, while the second complex (Mr 400,000) consisted of the heavy chain of factor Xa molecule and two subunits of alpha 2M. Factor Xa was able to form a bridge between two subunits of alpha 2M, either within one molecule of alpha 2M or by linking two molecules of alpha 2M. Complexes involving more than two molecules of alpha 2M were not formed.     

Structural arrangement in the alpha 2-macroglobulin--thrombin complex

The cysteine sulfhydryl groups of alpha 2-macroglobulin (alpha 2M) generated upon thrombin complex formation are in contact with the proteinase surface as evidenced by singlet--singlet energy transfer measurements from N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid-labeled thiol functions of alpha 2M to fluorescein isothiocyanate-labeled thrombin. The thrombin-alpha 2M binding is normally covalent, but the presence of hydroxylamine during the reaction leads to the formation of a non-covalent complex. The transfer energy determinations show that the alpha 2M binding sites of thrombin are quite similar, whatever covalent or non-covalent binding occurs.     

The reaction between diethylpyrocarbonate and sulfhydryl groups in carbocylate buffers.

Diethylpyrocarbonate reacts with sulfhydryl groups in the presence of carboxylate buffers to form a product which absorbs at 242 nm. The product is believed to be a thiol ester formed from the sulfhydryl compound and the buffer anion. This reaction interferes with the use of diethylpyrocarbonate to determine protein histidine residues when the reaction is performed in carboxylate buffers.     

Binding of IL-1 beta to alpha-macroglobulins and release by thioredoxin.

Human alpha 2-macroglobulin (H alpha 2M) is a major IL-1 beta binding plasma protein. The characteristics of the H alpha 2M IL-1 beta complex formation suggested, that cleavage of the internal thiol ester in other members of the alpha-macroglobulin family (alpha M) could enable these proteins to bind IL-1 beta. Characterization of optimal conditions for binding 125I IL-1 beta to H alpha 2M showed that H alpha 2M-IL-1 beta complex formation could be obtained over a pH range of 6.3 to 9 in the presence of some metal cations (i.e., Zn2+, Cd2+, Cu2+, Ni2+). Other divalent metal cations (i.e., Mn2+, Mg2+, Ca2+) were without effect. Time kinetic studies showed that binding of IL-1 beta to H alpha 2M was complete within 200 min and that H alpha 2M-IL-1 beta complexes became increasingly resistant to dissociation by boiling in SDS as a function of incubation time. Human pregnancy zone protein, rat alpha 1-, alpha 2-macroglobulin (R alpha 1M, R alpha 2M), all homologous with H alpha 2M, were tested for their ability to bind IL-1 beta. In each instance, alpha M-IL-1 beta complex formation was observed only after treatment of alpha M with methylamine, a primary amine that causes cleavage of the internal thiol ester in alpha M and the appearance of free thiol groups. Similarly, for each of these proteins, complex formation was increased several fold in the presence of Zn2+. Competition experiments using cytokines or proteins of similar molecular mass as IL-1 beta established that only unlabeled IL-1 beta was effective in inhibiting binding of 125I IL-1 beta to H"F" alpha 2M. Acylation of H"F" alpha 2M by diethylpyrocarbonate blocked the binding of IL-1 beta when analyzed by native PAGE. Deacylation of H"F" alpha 2M with hydroxylamine partially restored the binding capacity of H"F" alpha 2M further supporting the involvement of histidyl residues in the Zn2(+)-dependent binding of IL-1 beta. Reduced thioredoxin, but not its alkylated form, from Escherichia coli readily releases H"F" alpha 2M bound IL-1 beta under conditions that did not lead to reduction of disulfide bonds in H"F" alpha 2M. The action of thioredoxin also augmented IL-1-like activity in two independent bioassays suggesting that H"F" alpha 2M bound IL-1 beta is partially biologically inactive or latent. These results suggest that "activated" alpha M exert a modulating role for IL-1 beta by exposing specific binding sites, which are inaccessible in the native proteins.(ABSTRACT TRUNCATED AT 400 WORDS)