Time-resolved small-angle X-ray scattering experiment on association of isolated alpha- and beta-chains of human hemoglobin

The time course of the recombination of hemoglobin from isolated alpha- and beta-chains (human) was studied by following the change in small-angle scattering from a reaction mixture using synchrotron radiation. The scattering patterns were recorded successively within 51 s at intervals of 200 ms. The results suggest that when 2 mM solutions of CO-liganded alpha- and beta-chains are mixed in stoichiometric amounts at pH 7.4, the tetramerization process is a pseudo-first-order reaction with a rate constant of 0.059 s-1, i.e., a half-time of approx. 12 s.     

Genetic variation of haemoglobin alpha- and beta-chains in rabbits detected by isoelectric focusing and reversed-phase chromatography

Previous studies on the amino acid sequences and on the amino acid composition of peptides revealed genetic polymorphism both of the haemoglobin alpha-chain (Hb alpha) and beta-chain (Hb beta) in rabbits. In this study, rabbit haemolysates were analysed by isoelectric focusing in a narrow pH range (6.7-7.7) and by reversed-phase chromatography. Two variants were found for both Hb alpha and Hb beta. The two methods detected the same variants in this material. Inheritance data were consistent with the hypothesis that the observed Hb alpha and Hb beta variants were each controlled by two codominant, autosomal alleles. Haemoglobin polymorphism appears to be frequent in domestic rabbits since both variants of each chain were observed in all the three breeds studied.     

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.     

Glycosaminoglycans mediate the coacervation of human tropoelastin through dominant charge interactions involving lysine side chains.

Following cellular secretion into the extracellular matrix, tropoelastin is transported, deposited, and cross-linked to make elastin. Assembly by coacervation was examined for an isoform of tropoelastin that lacks the hydrophilic domain encoded by exon 26A. It is equivalent to a naturally secreted form of tropoelastin and shows similar coacervation performance to its partner containing 26A, thereby generalizing the concept that splice form variants are able to coacervate under comparable conditions. This is optimal under physiological conditions of temperature, salt concentration, and pH. The proteins were examined for their ability to interact with extracellular matrix glycosaminoglycans. These negatively charged molecules interacted with positively charged lysine residues and promoted coacervation of tropoelastin in a temperature- and concentration-dependent manner. A testable model for elastin-glycosaminoglycan interactions is proposed, where tropoelastin deposition during elastogenesis is encouraged by local exposure to matrix glycosaminoglycans. Unmodified proteins are retained at approximately 3 microM dissociation constant. Following lysyl oxidase modification of tropoelastin lysine residues, they are released from glycosaminoglycan interactions, thereby permitting those residues to contribute to elastin cross-links.     

Studies on the reaction of isocyanides with haemproteins. I. Equilibria and kinetics of the binding to the isolated chains of human haemoglobin

The equilibrium and kinetics of a series of alkylisocyanides (methyl, ethyl, isopropyl and tertiary butyl) reacting with the isolated chains of human haemoglobin (both in their SH and p-mercuribenzoate forms) has been investigated; the results lead to the following conclusions.

1.

(a) The reactions resemble, in their general properties, those with other ligands, such as O₂ or CO; there is absence of homotropic interactions (n ~ 1), and of heterotropic interactions (no Bohr effect) and a much higher equilibrium constant (I) than for intact haemoglobin, reflected kinetically in a large increase in the “on” constant (í).     

Immunological properties of antibodies against Mg(2+)-ATPase from Acholeplasma laidlawii membranes.

In the present study, antibodies were raised against the Mg(2+)-ATPase and the immunological relationships between the enzyme and other ATPase from a variety of biological membranes were determined. The anti Mg(2+)-ATPase antiserum inhibited 85% of the enzyme activity from A. laidlawii membranes. We demonstrate a specific selectivity of Mg(2+)-ATPase antiserum for antigenic determinants of the A. laidlawii membranes. Immunoblot studies of A. laidlawii membrane peptides indicated labeling of five bands, 66KD, 49KD, 34KD, 26KD and 13KD, corresponding to five subunits of the ATPase in A. laidlawii membranes.     

Chemical properties of the N-termini of human haemoglobin.

The chemical properties, namely pK and reactivity, of the N-termini of oxyhaemoglobin and deoxyhaemoglobin toward acetic anhydride and 1-fluoro-2,4-dinitrobenzene (Dnp-F) were determined by the competitive-labelling approach [Kaplan, Stevenson & Hartley, (1971) Biochem. J. 124, 289-229; Duggleby & Kaplan (1975) Biochemistry 14, 5168-5175]. At physiological pH and temperature, the valine-1 alpha and valine-1-beta amino groups had unusually low pK values, but showed only minimal changes in their pK values on deoxygenation. Between pH 7.5 and pH 8.0 a deviation was observed in the pH-reactivity profiles and the apparent pK values became markedly pH-dependent. It was found that Dnp-F, but not acetic anhydride, had an abnormally high reactivity toward the N-termini. It is concluded that the valine-1 alpha and valine-1 beta N-termini make little or no contribution to the alkaline Bohr effect at physiological pH values. The high reactivity toward Dnp-F is attributed to an interaction or binding near the N-terminal region, and the discontinuity in the pH-reactivity profile at moderate alkaline pH values to a conformational change which alters the environment of these groups.     

Immunological properties of monoclonal antibodies to human and rat prolyl 4-hydroxylase

Monoclonal antibodies to human (8 clones) and rat (12 clones) prolyl 4-hydroxylase [EC 1.14.11.2] were prepared and characterized as regards subclass, subunit specificity, inhibition and crossreactivity. Among the antibodies to the human enzyme, four clones showed the IgG1 subclass, two IgA, one IgG2b, and one IgM. Four clones reacted with the alpha subunit of the enzyme, while the others reacted with the beta subunit. The enzymatic activity was inhibited by four clones. Five clones crossreacted with the rat enzyme. One clone inhibited the rat enzyme. Among the antibodies to the rat enzyme, seven clones showed the IgG1 subclass, four IgG2a and one IgG2b. Seven clones reacted with the alpha subunit, and four with the beta subunit. One reacted with neither subunit. The enzymatic activity was inhibited by seven clones. Seven clones crossreacted with the human enzyme. Three clones inhibited the human enzyme.     

Comparative studies on the structure of the light chains of human immunoglobulins. IV. Assignment of a subsubgroup

Amino acid sequence analysis was done on a human lambda Bence Jones protein NIG-64 with the major objective of determining the sequence of the variable region. Nineteen tryptic peptides covering 216 residues were isolated from the completely reduced and aminoethylated protein, and 17 of these were completely sequenced. These comprised the entire variable region and 11 from the constant region. For the remaining peptides covering the rest of the constant region, only partial sequences or the amino acid compositions were determined. All the tryptic peptides could be arranged in order on the basis of the above results and homology with other human lambda light chains of the same isotype. The sequence of the variable region of the protein is highly homologous with that of protein New of subgroup V lambda I as compared with other proteins of the same subgroup, suggesting that subgroup V lambda I may be further divided into subsubgroups, namely subsubgroups V lambda I-1 and V lambda I-2.     

Immunological characteristics of monoclonal antibodies against human carcinoembryonic antigen (CEA)

Four hybridomas secreting monoclonal antibodies (MAbs) of the IgG1 subclass against human carcinoembryonic antigen (CEA) were obtained from fusion of P3-NS1/1-Ag4 myeloma cells with splenic cells from mice immunized with purified CEA. None of the MAbs showed cross-reactivity to perchloric acid extractable antigens from the normal human colon by an inhibition radioimmunoassay. However, MAb C27 showed the highest affinity to CEA. The intensity of immunofluorescence staining of human colorectal cancer cells with MAb C27 correlates well to the cellular CEA content of cancer cells. LS174T showed the highest intensity of fluorescence (95%) while COLO320DM and COLO320HRS were the lowest (0.5%). None of the normal human organs - colon, lungs, liver, spleen or kidneys-showed positive staining by immunoperoxidase anti-peroxidase (PA) techniques, while tissues from colorectal carcinoma (CRC), gastric carcinoma, hepatoma and lung cancer gave a positive rate of 100% (30/30), 96.6% (28/29), 32.1% (9/28) and 82.1% (69/84) respectively. Results suggest that MAb C27 can be used in immunodetection and radiolocalization of colorectal carcinoma.     

Haem degradation in human haemoglobin in vitro. Separation of the contribution of the alpha- and beta-subunits.

As part of an investigation into whether alpha 1-foetoprotein (alpha 1-FP) plays the same transport role in foetal serum as albumin does in the adult, the binding properties of both proteins were compared with respect to the binding of a series of compounds known to be bound by albumin's specific drug-binding sites. The binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid by rat alpha 1-FP and serum albumin was studied by equilibrium dialysis at 4 degrees C. Rat alpha 1-FP was shown to have neither albumin's high-affinity site II (diazepam as marker) nor its site III (digitoxin and cholic acid as markers). High-affinity binding by alpha 1-FP was found for the specific markers (warfarin, phenylbutazone, azapropazone) of albumin's drug-binding site I. However, instead of albumin's one high-affinity site/molecule, a mean value of 0.5 site/molecule was obtained with rat alpha 1-FP. Charcoal treatment at neutral pH of rat serum albumin did not affect its measured binding properties, but treatment of the alpha 1-FP led to an increased affinity for warfarin, phenylbutazone and azapropazone without a change in the measured number of sites, indicating competition for binding at this site by (an) endogenous ligand(s). These results are discussed in terms of the structures of the two proteins and with respect to the physiological implications of the differences found.     

The biosynthesis and assembly of T cell receptor alpha- and beta-chains with the CD3 complex

The biosynthesis, processing, and assembly of the TCR alpha- and beta-chains with each other and with the CD3 complex were investigated on both cell surface positive (TCR+CD3-) and negative (TCR-CD3-) cell lines. The results indicate that 1) in cell surface TCR-CD3- cell lines (MOLT 3, CCRF-CEM), TCR-beta, but not alpha-chains are present intracellularly. TCR-beta-CD3 complexes are readily found in these cell lines, but no evidence for final processing or cell surface expression of such incomplete TCR-CD3 complexes is observed. 2) In the cell surface TCR+CD3+ cell line HPB-ALL, both alpha- and beta-chains are present intracellularly. Whereas non-glycosylated forms of TCR-beta chain can be detected, only more mature forms of TCR alpha-chains are detected indicating that the alpha-chains are more rapidly glycosylated than the beta-chains. 3) The large majority of the intracellular alpha- and beta-chains is not disulfide linked and a small fraction of these is associated with CD3. 4) Only small amounts of the total intracellular TCR chains are found as CD3-associated disulfide-linked alpha beta-heterodimers. 5) Final processing of TCR chains for cell surface expression takes place after formation of these TCR-alpha beta-CD3 complexes. Thus, both the TCR alpha- and beta-chains are over-produced and only relatively small amounts of these chains form CD3-associated heterodimers that are processed for cell surface expression. Analogous results were obtained with a non-leukemic CTL clone. Based on these observations, a model for the biosynthesis and assembly of the TCR-CD3 complex is presented.