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1.
The addition of an excess of C2H5N13C to myoglobin and human adult and fetal hemoglobins, gives three characteristic NMR spectra with new 13C resonances respectively at δ = ?10,56 ppm, δ = ?7,03 and ?7,95 ppm and δ = ?6,28 and ?7,95 ppm (CH3CO2Na as external standard). These signals correspond to the C2H5N13C bound to the Fe(II) of the different heme units, according to CO exchange experiments. Characteristic resonances can be assigned to C2H5N13C bound to α, β and γ subunits. C2H5N13C appears as a more sensitive probe than 13CO for hemoprotein NMR studies.  相似文献   

2.
The principal component of normal adult human hemoglobin Ao, was equilibrated under various conditions with 13CO2. In addition, derivatives containing specifically carbamylated NH2-terinal groups in alpha or beta chains, or both, were prepared by treatment with cyanate, and equilibrated likewise to allow the identification of specific resonances observed by 13C nuclear magnetic resonance. In deoxyhemoglobin, a resonanance at 29.2 ppm upfield of external CS2 was assigned to the alpha chain terminal adduct, and one at 29.8 ppm to the beta chain terminal adduct. In the liganded state as the CO derivative, the terminal adduct on both chains showed a common resonance position at 29.8 ppm. Small effects of pH on the resonance positions were observed. Under certain conditions, a resonance was observed at 33.4 ppm, probably not ascribable to a carbamino compound. A carbamino resonance that became prominent at higher pH was found at 28.4 ppm, and is tentatively ascribed to one or more adducts on epsilon amino groups. The beta chain resonances in particular are minimized by the presence of inositol hexaphosphate or 2,3-diphosphoglycerate. Quantitative analysis of the resonance intensities shows that the effects of conversion from the deoxy to the liganded state in reducing the degree of carbamino adduct is much more pronounced for the beta than for the alpha chains.  相似文献   

3.
The abnormal hemoglobin Zurich (β63 his→arg) exhibits abnormal properties. Thus, νCO occurs at 1951 cm?1 for HbACO while HbZCO shows bands at 1950 cm?1 and 1958 cm?1 for CO bound in α and β chains respectively (the βCOs are displaced less readily by O2). Acid catalyzed reductive displacement of superoxide by azide is slower on the β chain of HbZO2 than on the α chain under conditions where with HbAO2 both chains appear equally reactive. The one electron donor hydroquinone produces metHb and peroxide more rapidly from HbZO2 than from HbAO2. These property differences can be related to the β63 residue. Such studies provide generally useful probes of the structural basis for hemoglobin diseases.  相似文献   

4.
The proton nmr spectra of the synthetic valency hybrids, α2+CN)2, (α+CN)2β2 of hemoglobin A and the natural valency hybrids of the mutant hemoglobins Boston, Iwate and Milwaukee have led to the unambiguous assignment of the two proximal histidyl imidazole exchangeable proton signals at 64 and 76 ppm to individual α and β subunits, respectively. New single non-exchangeable proton resonances detected in the extreme downfield region of the spectra of Hbs Boston and Iwate are tentatively assigned to the coordinated tyrosine of the mutated α chains.  相似文献   

5.
The 15N paramagnetic shifts of iron-bound C15N? were studied for myoglobin, hemoglobin, cytochrome c and other modified hemoproteins. Two characteristic 15N resonances at 977 and 1045 ppm (with respect to 15NO3? as an internal standard) were found for human adult hemoglobin cyanide, while only single resonances were observed for other cyano hemoproteins. These two resonances are assigned to iron-bound C15N of α and β subunits of hemoglobin. The substantial difference in the C15N isotropic shifts in various hemoproteins is discussed in relation to iron-proximal histidine binding and heme-apoprotein interactions.  相似文献   

6.
Hemoglobin MSaskatoon (α2Aβ263tyr) has two α chains in the normal ferrous state, while its two β chains are in the ferric state. The reaction of hemoglobin MSaskatoon with carbon monoxide at pH 7 and 20 °C in the presence and absence of dithionite was studied. In the absence of dithionite only the α chains react and the combination rate is slow and similar to that of normal deoxyhemoglobin. After the addition of dithionite the rate of reaction is greatly increased initially and then decreases to a rate similar to that seen in the absence of dithionite. The dissociation of oxygen from hemoglobin MSaskatoon at pH 7 and 20 °C was found for the α subunits to be similar to that seen for normal oxyhemoglobin. This similarity in the kinetic properties of normal hemoglobin and the α subunits of hemoglobin MSaskatoon in both ligand combination and dissociation reactions indicates that the α subunits of hemoglobin MSaskatoon undergo a structural transition from a low to high affinity form on liganding. Since the β subunits react rapidly with carbon monoxide even when the α subunits are unliganded, it appears that the ligand binding sites of the β chains are uncoupled from the state of liganding of the α subunits.  相似文献   

7.
A procedure to separate the α and β globin chains of rabbit hemoglobin, denatured with sodium dodecyl sulfate in the presence of mercaptoethanol, on a column of polyacrylamide gel was developed. The identity of the two separated chains was verified by (a) differences in distribution of radioactivity between the chains when the hemoglobin samples were labeled uniformly with various 3H- or 14C-labeled amino acids; (b) the analysis of the chain distribution of radioactivity in purified hemoglobin isolated from rabbit reticulocytes, pulse-labeled with [3H] leucine; and (c) the separation pattern of a mixture of authentic [α-3H]- and [β-14C]-labeled globin chains. The globin chains of human hemoglobin A also could be separated in a similar manner. This procedure is particularly useful when only microgram quantities of hemoglobin are available for study.  相似文献   

8.
9.
The orientation of melittin in lecithin membranes was investigated by means of 13C-NMR spectroscopy. Phospholipase-free melittin was labeled with 13C-methyl groups at the -amino side chains of lysine 7, 21, and 23. From the pH dependence of the corresponding 13C resonances, pK values of the lysine residues were derived that were different for free and membrane-bound melittin. The shift reagent Pr(NO3)3 induced shifts in the 13C resonance position of all three lysines when melittin and the shift reagent were added to a lecithin vesicle suspension, whereas Pr3+ ions included in the inner volume of the vesicles did not affect the 13C resonances of melittin bound to the outer vesicle membrane. A wedge-like structure was derived for the membrane-bound melittin, the lysine side chains of which are freely accessible to the aqueous solvent.Abbreviation NOE Nuclear Overhauser Enhancement  相似文献   

10.
The heme iron of the β chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the β globin chain. The presence of freely reactive -SH groups on the α chains of cat and sheep hemoglobins does not alter the course of this reaction: only the β hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two β chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, α2Humanβ2Cat and α2Catβ2Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the β subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid α2Catβ2Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or α2Humanβ2Cat. Cat β globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2β. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the β chain and is associated with histidine 2β.  相似文献   

11.
The binding of oxygen to hemoglobin has been investigated by 19F-nuclear magnetic resonance spectroscopy. The 19F-nmr spectrum of hemoglobin trifluoroacetonylated at cysteine β 93 exhibits chemical shift changes on binding of ligands, which differ depending on which chains are undergoing complexation. Comparison of these changes to the fractional ligation of all chains, determined concurrently from the fractional change in the visible spectrum, shows that initial oxygen molecules bind preferentially to α-chains. The 19F-nmr spectrum of partially oxygenated hemoglobin contains resonances at the normal chemical shift positions of the oxygenated and deoxy species, in addition to two small resonances at intermediate positions. Analysis of the relativ magnitudes of these four peaks as functions of oxygen pressure permits identification of the intermediate species  相似文献   

12.
The 13C-NMR spectrum at 90.5 MHz has been obtained for the photosynthetic thylakoid membrane of spinach. Specific lipid and chlorophyll resonances can be assigned in the high resolution spectrum, although protein resonances are not observed. It can be estimated from resonance intensities that at least 30% of the plant chlorophyll contributes to the high resolution 13C spectrum with the remainder broadened by incomplete motional averaging. The resonance linewidths of the observed chlorophyll phytol chains are approximately the same as those of the lipid hydrocarbon chains, indicating a similar motional state and suggesting that this particular pool of chlorophyll is lipidbound or at most only loosely associated with proteins.  相似文献   

13.
Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P50) and 1.96 (n value), and a small Bohr effect (δH+ = ?0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O2 affinity at P50 of 2.5 mmHg which may assure efficient utilization of the lung O2 reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl2·6H2O] solution at a pH of 7.0–8.5.  相似文献   

14.
15.
The hemoglobin of the sea snakeMicrocephalophis gracilis was purified and the primary structure of the α and β chains determined. This is the first sea snake hemoglobin structure characterized, and apparently also the first complete structure of any snake hemoglobin (an α chain of a viper was known), allowing judgments of reptilian variants. Variations between the sea snake form and other reptilian forms are large (52–65 differences for the α chains), of similar order as those between the sea snake and avian (56–65 differences) or human (58 differences) forms. Functionally, 19 residues at α/β contact areas and 7 at heme contacts are exchanged in relation to the human α and β chains. Four positions of the sea snake hemoglobin contain residues thus far unique to this form. However, all replacements appear compatible with conserved overall functional properties.  相似文献   

16.
Solvent-proton longitudinal magnetic relaxation rates as dependent on temperature were measured for human (H)/canine (C) valency hybrids of the type {αH(III)βC(II)}2 and {αC(II)βH(III)}2. The two metheme irons in the human methemoglobin chains induce quite different proton magnetic relaxation (pmr) rates reflecting a tighter β-heme-pocket compared to the α subunit. Both heme-pockets appear to be loosened in the presence of inositol hexaphosphate (IHP) although this allosteric effector binds only to the β chains, the binding assumed to be the same for canine as for human hemoglobin. The subunit nonequivalence is retained also in the T-quaternary state induced by IHP. In the species hybrids the pmr rates due to the metheme iron are sensitive to the valency (ligand) state, which was either CO or H2O in the partner half of the hybrid. All results show very clearly the interrelationship of the tertiary (protomer) structure with the quaternary (oligomer) structure in hemoglobin.  相似文献   

17.
The ratio of the apparent rates of ligand binding to the α and β subunits of human hemoglobin on mixing with non-saturating amounts of nitric oxide has been measured by two independent methods. Electron spin resonance measurements permit direct determination of the ratio of the amounts of the respective chains bound by NO. In stopped-flow kinetics measurements, use was made of the known difference in the kinetic constants of α and β chains in hemoglobin in the reaction with n-butyl isocyanide. Both methods concur in indicating that the apparent association rate constant of NO is greater for the α than for the β chain.  相似文献   

18.
The human AKAP13 protein contains DH and PH domains, which are responsible for its cell transforming activity. Despite its biomedical importance, the contribution of the PH domain to AKAP13 activity remains unclear and no three dimensional structure is available to date. Here we report the backbone and side chain 1H, 13C and 15N resonance assignments of a 20 kDa construct comprising the uniformly 13C and 15N labeled AKAP13-PH domain and an associated helix from the DH domain which is required for its stable expression. Resonance assignment has been achieved using conventional triple resonance experiments; 95% of all back bone resonances and more than 90% of side chain resonances have been successfully assigned. The 1H, 13C and 15N chemical shifts have been deposited in BMRB with accession number of 16195.  相似文献   

19.
The vicinal coupling constants 13C′-13Cγ were measured in aspartic acid and phenylalanine (85 % 13C enrichment) as free amino acids and in the peptides Asp-Pro and Gly-Pro-Phe. These coupling constants used in connection with those between the α -and the β-protons provide the unambiguous assignment of rotamers I and II in the Asp and Phe side chains. The method is generally applicable to other amino acids and residues even in large peptides. A possible set of Jgc,c and Jtc,c values is proposed for the use of carbon 13-carbon 13 vicinal coupling constants in the side chain conformational studies of amino acid residues with a free carboxyl group.  相似文献   

20.
The 13C chemical shifts and spin-lattice relaxation times are reported for cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu). The chemical shifts of the D and L leucyl residues in the cyclic peptides differ from each other by 1.8 and 3.6 parts per million for the α and β carbons, respectively. The α-carbons of the prolyl residues differ by 1.0 ppm as a consequence of proximity to a D or an L leucyl residue. The 13C spin-lattic relaxation time(T1) of the prolyl residues, but not the leucyl residues, in both compounds are indicative of difference in conformational equilibria within the pyrrolidine ring in the L -L isomer as compared to the L -D isomer. Anisotropic overall molecular reorientation is not responsible for the differences observed in the T1 values. The differences in T1 values and chemical shifts between cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu) appear to result from a difference in conformations of the two diketopiperazine rings.  相似文献   

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