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A new method of coupling proteins to insoluble polymers 总被引:1,自引:0,他引:1
P V Sundaram 《Biochemical and biophysical research communications》1974,61(2):717-722
The A chain isolated from biologically active human thrombin is thirteen residues shorter from the amino terminus than the A chain isolated from bovine thrombin. Automated Edman degradation of the reduced, alkylated A chain permitted the direct identification of 34 out of the 36 residues and established all tryptic overlaps. The NH2-terminal residue is threonine. This residue is homologous to threonine-14 of the A chain of bovine thrombin. Human thrombin A chain has one half cystine linking it to the larger B chain. Methionine, histidine, valine and tryptophan are not present. There are nine acid residues (Glu, Asp) but none of their respective amides. 相似文献
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Observation of small conformational changes in the sperm-whale myoglobin by the far-infrared spectra
Small conformational changes in a molecule of sperm-whale myoglobin in its native solid state for different pH values at room temperature as well as during heat denaturation in alkali medium at different stages of unfolding of the globule were observed by using far-infrared spectroscopy in the region from 30 to 600 cm?1. The changes appeared in the absorption bands near 420 and 470 cm?1 ascribed to the side-chain vibrations of helical segments of the myoglobin molecule. For the first time the high structural sensitivity of the far-infrared region of the skeletal vibrations has been confirmed experimentally and the applicability of this technique to globular proteins demonstrated. 相似文献
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1. Sperm-whale apomyoglobin was digested with chymotrypsin in a dialysis sac. The ultrafiltrate contained incompletely hydrolysed fragments which partially inhibited the precipitation of metmyoglobin and apomyoglobin by some antisera produced against metmyoglobin. The inhibitory activity was stable to heating at 100 degrees and depended on the peptide structure. 2. The fragments were fractionated according to molecular size and were purified by ion-exchange chromatography. Six pure peptides and two peptides which contained a minor impurity were isolated. Their amino acid compositions and N-terminal amino acid sequences were determined and their entire amino acid sequences deduced from the known amino acid sequence of sperm-whale myoglobin. 3. The peptides formed no detectable precipitates with the antisera. Five of the eight peptides partially inhibited the precipitation of apomyoglobin and/or metmyoglobin by one antiserum. Six of the peptides inhibited the precipitation of apomyoglobin by one or other of two antisera; at least two of these peptides inhibited both antisera. One peptide failed to inhibit the precipitation of either antigen by either antiserum. Two of the peptides possessed the same serological specificity. 4. The molar ratios of inhibitors to antigen for 50% of the maximum inhibition decreased as the molecular size of the inhibitor increased. With one antiserum and with apomyoglobin as the antigen, molar ratios 12 and 80 were obtained for peptides with molecular weights 2051 and 793 respectively. 5. The size and structure of an antigenic site is discussed in relation to the known steric configuration of myoglobin. 相似文献
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Immunochemistry of sperm-whale myoglobins prepared with various modified porphyrins and metalloporphyrins 总被引:7,自引:6,他引:1
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M. Z. Atassi 《The Biochemical journal》1967,103(1):29-35
1. The preparation and characterization of manganese, iron, cobalt, nickel, copper and zinc metalloporphyrins is described. Ferrihaem was also esterified with pyrid-4-ylpropanol and the derivative characterized as the diester. 2. Complexes of these various porphyrins, as well as protoporphyrin IX, with apomyoglobin were formed and the resulting artificial myoglobins characterized. 3. Very little complex-formation was obtained with nickel, cobalt and manganese metalloporphyrins and apomyoglobin. 4. Myoglobin prepared with copper metalloporphyrin was immunochemically identical with native ferrimyoglobin. All the other artificial myoglobins were less reactive to varying degrees. 5. The changes in antigenic reactivities were attributed to conformational reorganization caused by the different co-ordination tendencies of the various metals or by the modification of the side chains of the haem. 相似文献
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Prediction and conformation by synthesis of two antigenic sites in human haemoglobin by extrapolation from the known antigenic structure of sperm-whale myoglobin.
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The complete antigenic structure of sperm-whale myoglobin was previously determined in our laboratory. By structural analogy with myoglobin, two regions in human haemoglobin were predicted to comprise antigenic sites. One region was on the alpha-chain [alpha-(15-23)] and the other on the beta-chain [beta-(16-23)]. These two regions were synthesized, purified and characterized, and their immunochemistry was studied. Each peptide was able specifically to bind considerable amounts of haemoglobin antibodies. In a set of homologous proteins, barring any drastic conformational or electrostatic inductive effects exerted by the substitutions, and allowing for obstruction due to subunit interaction, the determination of the antigenic structure of one protein may serve as a useful starting model for the others. 相似文献
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Hydroxypropyl cellulose (HPC) was used as a core molecule for controlled grafting of monomers by ATRP, the aim being to produce densely grafted comb polymers. HPC was either allowed to react with an ATRP initiator or the first generation initiator-functionalized 2,2-bis(methylol)propionic acid dendron to create macroinitiators having high degrees of functionality. The macroinitiators were then "grafted from" using ATRP of methyl methacrylate (MMA) or hexadecyl methacrylate. Block copolymers were obtained by chain extending PMMA-grafted HPCs via the ATRP of tert-butyl acrylate. Subsequent selective acidolysis of the tert-butyl ester moieties was performed to form a block of poly(acrylic acid) resulting in amphiphilic block copolymer grafts. The graft copolymers were characterized by 1H NMR and FT-IR spectroscopies, DSC, TGA, rheological measurements, DLS, and tapping mode AFM on samples spin coated upon mica. It was found that the comb (co)polymers were in the nanometer size range and that the dendronization had an interesting effect on the rheological properties. 相似文献
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A criterion has been devised for the assessment of intermolecular contacts between protein subunits coupled to a gel matrix. After reversible coupling by way of disulfide groups to Sepharose 4B, the system is exposed to a bifunctional reagent. The protein is then released by reduction, and the proportion of dimers and higher oligomers formed by cross-linking is determined by gel electrophoresis, followed by densitometry of the stained gels. Experiments were performed with G-actin coupled to dithio-2-dipyridyl Sepharose 4B. At low concentration of coupled protein, no species other than the monomer were obtained; under these conditions any intermolecularly cross-linked species would represent pre-existing associated states coupled to the matrix. At higher protein concentrations dimers and higher species progressively appear. Their proportion is much higher than predicted on the basis of a random statistical distribution of coupled molecules throughout the accessible internal volume of the matrix. It follows that protein-protein contacts can occur either because of high flexibility of the polysaccharide chains of the matrix, or because the protein is partly (but not wholly) present as a shell on the surface of the beads. With Affi-gel 10, which has N-hydroxysuccinimide ester coupling groups, similar experiments were performed, taking advantage of the degradation of the matrix under relatively mild acid conditions. In this case the degree of cross-linking was much lower than in the Sepharose 4B system at the same protein concentration. However, this medium proved unsatisfactory for the measurement of interactions of the bound actin with other muscle proteins present in the mobile phase. The results with Sepharose 4B support the validity of previous studies on interaction of monomeric actin with other muscle proteins. 相似文献
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Haemophilus influenzae has an absolute growth requirement for heme. One potential in vivo source of heme is the protein myoglobin which is found at low levels in human serum. No tested H. influenzae strain was able to use myoglobin as a heme source. However, all strains were able to utilize the heme from myoglobin when myoglobin was complexed with haptoglobin. Utilization of the haptoglobin-myoglobin complex was shown to be mediated by the previously described hemoglobin/hemoglobin-haptoglobin-binding proteins of H. influenzae. 相似文献