The alpha 1 beta 1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen.

When the alpha and beta chains were separated from human oxyhemoglobin (HbO(2)), each individual chain was oxidized easily to the ferric form, their rates being almost the same with a very strong acid-catalysis. In the HbO(2) tetramer, on the other hand, both chains become considerably resistant to autoxidation over a wide range of pH values (pH 5-11). Moreover, HbA showed a biphasic autoxidation curve containing the two rate constants, i.e. k(f) for the fast oxidation due to the alpha chains, and k(s) for the slow oxidation to the beta chains. The k(f)/k(s) ratio increased from 3.2 at pH 7.5-7.3 at pH 5.8, but became 1 : 1 at pH values higher than 8.5. In the present work, we used the valency hybrid tetramers such as (alpha(3+))2(beta O(2))(2) and (alpha O(2)(2)(beta(3+))(2), and demonstrated that the autoxidation rate of either the alpha or beta chains (when O2- ligated) is independent of the valency state of the corresponding counterpart chains. From these results, we have concluded that the formation of the alpha 1 beta 1 or alpha 2 beta 2 contact suppresses remarkably the autoxidation rate of the beta chain and thus plays a key role in stabilizing the HbO(2) tetramer. Its mechanistic details were also given in terms of a nucleophilic displacement of O(2)(-) from the FeO(2) center, and the emphasis was placed on the proton-catalyzed process performed by the distal histidine residue.     

Oxygen equilibria of hybrid-heme hemoglobins containing proto- and mesoheme groups. On the nonequivalence of alpha and beta chains.

Hybrid-heme hemoglobins, alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2, were prepared, and the O2 equilibria of their alpha and beta chains were measured separately at the isosbestic points of the partner chains at different pH values and in the presence and absence of inositol hexaphosphate. The Adair equation was extended to distinguish between the O2 saturations of the alpha and beta chains, and the seven equilibrium parameters were obtained by curve fitting to those equations. The results showed that the beta chains have an affinity slightly higher than the alpha chains in the binding of the first O2 molecule. For the second O2 molecule, the molecular species that has been oxygenated on the alpha chain has a higher affinity than that carrying O2 on the beta chain. The slopes of the Hill plots were higher for the alpha chain. The O2 saturation curves for the alpha and beta chains were calculated from the parameters averaged for the hybrids alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2 in order to cancel the effects of the heme replacement. The curves showed that the difference in O2 saturation between the two kinds of chains depends on the conditions and on the degree of O2 saturation. It was concluded that the functional difference between the chains is small enough so that it is not required to modify the models already accepted for the cooperativity of hemoglobin.     

Binding of human hemoglobin and its polypeptide chains with haptoglobin coupled to an agarose matrix.

The interactions of human haptoglobin covalently linked to agarose with human hemoglobin and with p-chloromercuribenzoic-acid-treated alpha and beta chains (alpha* and beta* chains) were studied by flow chromatography and equilibrium binding. The results indicate that in solid state, haptoglobin maintains the same binding characteristics as in solution, the order of binding affinities being: hemoglobin greater than alpha* chain greater than beta* chain. The study of the binding parameters of the alpha* chain shows an heterogeneity of binding sites on the haptoglobin and an average affinity constant Ka of 3.6 X 10(4)l/mol.     

The amino acid sequences of the alpha and beta chains of hemoglobin from the snake, Liophis miliaris

The hemoglobin of Liophis miliaris has unusual properties. The hemoglobin is dimeric in the oxy form, and the cooperativity of O2 binding is very low, but both the Bohr effect and cooperativity are greatly enhanced in the presence of ATP (Matsuura, M. S. A., Ogo, S. H., and Focesi, A., Jr. (1987) Comp. Biochem. Physiol. 86A, 683-687). Four unique chains (2 alpha, 2 beta) can be isolated from the hemolysate. The amino acid sequences of one alpha and one beta chain have been determined in an effort to understand the functional properties. Comparison of the sequences with those of the alpha and beta chains of human Hb shows the following. (i) All 7 of the residues in the beta chain normally conserved in globins are identical to those of the human chain: Gly(B6), Phe(CD1), His(E7), Leu(F4), His(F8), Lys(H10), and Tyr(HC2), except that the distal His(E7) has been replaced by Gln in the alpha chain. (ii) All heme contact residues in the beta chain are identical with those in the human chain, but two differences are present in the alpha chain: the distal His(E7) is replaced by Gln and Met(B13) by Leu. (iii) All residues that form the binding site for organic phosphates are identical to those in human Hb. (iv) The major residues that contribute to the normal Bohr effect in human Hb, Asp-beta 94, His-beta 146, and Val-alpha 1 are conserved. (v) All beta chain residues at the alpha 1 beta 2 interface are identical with those in the human chain except two: Glu(G3)----Val and Glu(CD2)----Thr; these differences in charged residues may explain the dissociation to dimers. (vi) The 23 residues of the alpha chain in the alpha 1 beta 2 contact region are identical with those of the human chain except three: Phe(B14)----Leu, Thr(C3)----Gln and Pro(CD2)----Ser. (vii) A total of 17 differences occur at the alpha 1 beta 1 interface, 11 in the alpha chain and 6 in the beta chain.     

Overproduction of alpha chains provides a proton-insensitive component to the bluefish hemoglobin system

Expression of alpha and beta chains and their post-translational assembly into alpha(2)beta(2) tetramers is fundamental to the formation and function of most vertebrate hemoglobins. There is a strong evolutionary bias that favors expression of equal amounts of the two types of chains, because cooperativity, pH sensitivity, and anionic control of function occurs only for the alpha(2)beta(2) tetramers. Remarkably, an over-production of alpha chains, as in the pathological condition known as beta thalassemia in humans, is adaptive rather than pathological in the bluefish hemoglobin system. The thalassemia of the bluefish is a novel means of providing for oxygen uptake and delivery when low pH conditions incapacitate the highly pH-sensitive Root effect hemoglobins of the fish. Although fish often have pH-insensitive along with highly pH-sensitive hemoglobins, having pH-insensitive alpha chain monomers in circulation is an unusual structural variation. The role of bluefish alpha chains in oxygen transport is enabled by their remarkably lower oxygen affinity relative to human alpha chains. This is the first reported case of a thalassemic condition that is maintained in a species as an adaptive advantage.     

Allosteric interpretation of the oxygen-binding reaction of human hemoglobin tetramers

An allosteric model is presented that provides a simple explanation for the low population of triply ligated species, relative to the other species, in the oxygenation of human hemoglobin tetramers as found in high-concentration studies [Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry (preceding paper in this issue)]. The model is a quantitative interpretation of the Perutz mechanism [Perutz, M. F. (1970) Nature (London) 228, 726-739] and is based on a number of structural and thermodynamic findings so far reported in the analysis of hemoglobin properties. Human hemoglobin is assumed to exist in two quaternary states: the T or low-affinity state and the R or high-affinity state. An extreme chain heterogeneity in the T state is postulated so that oxygen binds only to the alpha chains. Nearest-neighbor interactions between the alpha chains may lead to cooperativity within the T state. The R state is noncooperative, and both the alpha and beta chains have equal oxygen affinity.     

Differential expression of laminin chains in the human major salivary gland

Laminin represents a macromolecule family. The heterotrimeric isoforms of laminin can be determined by immunohistochemical demonstration of the single chains. The laminin chain heterogeneity of the basement membrane in adult human major salivary glands was evaluated in relation to cellular differentiation of the epithelia and the stromal compartment. Monoclonal antibodies to the laminin alpha1, alpha3 (BM165) chains and epiligrin reacted with the basement membranes of serous and mucous acini and of intercalated, striated and excretory ducts. As evidenced by a double-labelling technique, the alpha2 chain showed a spatial association with the myoepithelium of the acini, whereas the ductal basement membranes containing no myoepithelial cells were negative. Almost exclusively, beta1 chain was detected in acinar basement membrane, beta2 chain whereas in ductal basement membrane. gamma2 chain is a unique chain belonging to the laminin-5 isoform. It was restricted to the ductal basement membrane. alpha1, alpha2, beta1, beta2 and gamma1 chains were detected in nerves of salivary tissue and alpha1, alpha3, beta1, beta2 and gamma1 chains and epiligrin in blood vessels. Our results indicate that the acinar ductal unit contains basement membranes with different isoforms, which relate to cell differentiation and cell function. © 1998 Chapman & Hall     

Double-mixing kinetic studies of the reactions of monoliganded species of hemoglobin: alpha 2(CO)1 beta 2 and alpha 2 beta 2(CO)1.

The kinetics of CO association to and dissociation from the two isomers of monoliganded species alpha ICO beta I(alpha II beta II) and alpha I beta I (alpha II beta COII) has been studied by double-mixing stopped-flow and microperoxidase methods. The monoliganded species were generated by hybridization between excess ferric Hb and alpha CO2 beta +2 or alpha +2 beta CO2 prepared by high-pressure liquid chromatography (HPLC). The results indicated that: 1) there were no significant differences in the reactivities of alpha and beta chains in the first step of ligation; 2) in the second step of ligation there was significant cooperativity in the reaction of deoxyhemoglobin with 0.05 or 0.1 equivalent of CO. Diliganded species were therefore formed in significant amounts. The double-mixing HPLC results suggested that in the second step of ligation alpha chains reacted faster than the beta chains, and the main diliganded species formed was alpha I beta ICO (alpha IICO beta II) or its isomer alpha ICO I(alpha II beta IICO). These results seem to indicate that the reaction of the first CO is mostly random and in the second step of ligation CO binds more to the tetramers in which one beta chain is already ligated: alpha I beta I (alpha II beta II) + CO----alpha ICO beta I (alpha II beta II) and alpha I beta ICO (alpha II beta II) + CO----alpha I beta ICO (alpha IICO beta II).     

Interaction of hemoglobin and its component alpha and beta chains with band 3 protein

The equilibrium binding of hemoglobin to isolated band 3 protein exhibited positive cooperativity [Hill coefficient = 1.65 +/- 0.1; total number of binding sites at pH 6.6 in 5 mM sodium phosphate buffer = 32 500 +/- 940 pmol/mg; Ka = (3.0 +/- 0.5) X 10(5) M-1]. The binding was reversible and ionic in nature as the bound hemoglobin was readily displaced by KCl, ATP, and 2,3-diphosphoglycerate, the latter two being more effective than KCl on a molar basis. The ratio of the interaction of hemoglobin to band 3 protein per se was 1:1, whereas the band 3 preparation as a whole (protein + lipids) was 3:1. Saturating levels of glyceraldehyde-3-phosphate dehydrogenase blocked only 33% of the total binding sites which were localized at the cytoplasmic segment; the remaining 67% was localized in lipids by their extraction with acetone. Reconstitution of acetone-extracted band 3 with phospholipid liposomes indicated phosphatidylserine as the binding site. The positive cooperativity in binding to acetone-extracted band 3 was increased (Hill constant = 2.1 +/- 0.1) compared to the band 3 preparation. After separation of the alpha and beta chains of hemoglobin, only the alpha chain binds to band 3 with positive cooperativity to an extent of 45-50% of native hemoglobin with similar affinity. The binding capacity of p-(hydroxymercuri)benzoate (HMB) derivatives of hemoglobin and its alpha chain was less than that of native hemoglobin, whereas HMB-beta chain or beta chain did not bind.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin produced in choriocarcinoma. Appearance of triantennary sugar chains and unique biantennary sugar chains

Human chorionic gonadotropin (hCG) highly purified from urine of the patient with choriocarcinoma contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from polypeptide portion by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The structures of these sugar chains were determined by the combination of sequential glycosidase digestion, periodate oxidation, and methylation analysis. As compared with the sugar chains of normal urinary and placental hCG reported previously, they include several prominent structural differences. More than 97% of the sugar chains of choriocarcinoma hCG was free from sialic acid, while the sugar chains of normal hCG were mostly sialylated. Choriocarcinoma hCG contains unusual biantennary complex-type sugar chains in addition to regular tri-, bi-, and monoantennary sugar chains. These sugar chains have two outer chains linked at the C-2 and C-4 positions of the same alpha-mannosyl residue of the trimannosyl core. Since normal hCG does not contain any triantennary sugar chains, occurrence of Gal beta 1 leads to 4GlcNAc beta 1 leads to 4Man alpha 1 leads to group is another characteristic feature of the sugar chains of choriocarcinoma hCG. The evidence that the monoantennary sugar chain of Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc leads to Asn is not found in normal hCG and the sum total of fucosylated sugar chains is 50%, which is twice as much as normal hCG, indicated that fucosylation is also modified in choriocarcinoma tissue.     

Oxidation-reduction potentials of human fetal hemoglobin and gamma chains. Effects of blocking sulfhydryl groups.

The oxidation-reduction equilibrium of the gamma chains of human fetal hemoglobin (Hb F) has been studied and compared with that of the alpha and beta chains of human adult hemoglobin (Hb A). The effects of the sulfhydryl (--SH) reagents, iodoacetate, iodoacetamide, and p-mercuribenzoate (PMB), on the three kinds of chains and on Hb F have been compared. The midpoint potentials (E-m) of all three sorts of chains are lower than those of tetrameric hemoglobin A or F. The E-m values of alpha chains are the lowest, E-m = 0.049 volt at 6 degrees, and are unaffected by pH change or by PMB treatment, at least from pH 6 to 8. The E-m values of beta-SH chains are higher; E-m = 0.102 volt at pH 7, decreasing to 0.050 volt at pH 8, both at 6 degrees. These results agree with those of Banerjee and Cassoly ((1969) J. Mol. Biol. 42, 337-349). They reported no effect of PMB on beta chains, but we find that 2 eq of PMB/chain raise E--M to 0.139 volt at pH 7 at 6 degrees, chiefly as the result of reaction at beta-93, not at beta-112. Carboxymethylation at beta-93 has an insignificant effect compared with that of PMB. The oxidation-reduction potential of gamma chains is similar to that of beta chains. E-m = 0.098 volt at pH 7 at 6 degrees, decreasing to 0.064 at pH 8 and 0.010 at pH 9. The effects of --SH reagents, reacting at position gamma-93 (the only --SH group present in gamma chains), are essentially the same as those seen with beta chains. The oxidation-reduction potential of Hb F is almost identical with that of Hb A, except for being 0.008 volt lower at pH 6 at 6 degrees. This agrees with the results reported by Flohe and Uehleke ((1966) Life Sci. 5, 1041-1045). PMB or iodoacetamide treatment lowers E-m by 0.02 to 0.03 volt, depending on the pH, from 6 to 9, in much the same way as previously reported for Hb A(Brunori, M., Taylor, J.F., Antonini, E., Wyman, J., and Rossi-Fanelli, A. (1967) J. Biol. Chem. 242, 2295-2300). The "residual oxidation Bohr effect" noted in Hb F can be attributed to the oxidation Bohr effect of the gamma chains. The apparent pK of the heme-linked water molecule was found at 25 degrees to be, for Hb F, 8.1; for gamma-SH chains, 7.85; for gamma-PMB chains, 8.35; and for gamma chains treated with iodoacetate, 7.80. Sedimentation coefficients, s-20, w, at a protein concentration of 5 mg/ml, were found to be, for fetal hemoglobin 4.09, for iodoacetamide-treated fetal hemoglobin 4.04, for PMB-treated fetal hemoglobin 3.41, for fetal gamma-SH chains 4.25, and for fetal gamma-PMB chains 3.08.     

Recombinant human laminin-5 domains. Effects of heterotrimerization, proteolytic processing, and N-glycosylation on alpha3beta1 integrin binding

Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The alpha3' chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type alpha3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, beta3'and gamma2', produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the alpha3' chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1-3, but not the single chains, supported binding of soluble alpha3beta1 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with alpha3beta1 integrin. The N-glycosylation sites in wild-type alpha3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor alpha3beta1 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4-5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the alpha3'LG1-3 chain showed an increased binding affinity for alpha3beta1 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with alpha3beta1 integrin.     

Mouse beta thalassemia, a model for the membrane defects of erythrocytes in the human disease

The present study describes the pathophysiology, at the cellular level, of the mouse beta thalassemia and shows the pertinence of this model for the human disease. The homozygous state of mouse beta thalassemia is characterized by a clinical syndrome similar to the human beta thalassemia intermedia, but it cannot be explained by the small deficiency in beta chain synthesis. The small pool of unpaired and soluble alpha chains present in mouse reticulocytes contrasts with the large amount of insoluble alpha chains in erythrocytes which is induced by the high instability of mouse alpha chains and the absence of significant proteolysis. The amount of insoluble alpha chains associated with red cell ghosts is similar in human and mouse disease of similar severity. The study of membrane protein defects showed a decreased amount of spectrin (alpha and beta chains) and dramatic changes in the distribution of the most reactive thiol groups of membrane proteins. These results were similar to that previously described in the human disease (Rouyer-Fessard, P., Garel, M. C., Domenget, C., Guetarni, D., Bachir, D., Colonna, P., and Beuzard, Y. (1989) J. Biol. Chem. 264, 19092-19098). Abnormal density distribution curves of erythrocytes and oxidant-induced lysis of red blood cells used as functional tests were similar in the human and mouse beta thalessemia. We conclude from the present study that 1) mouse beta thalassemia is an excellent model for the membrane defects occurring in the human disease; 2) disease expression is not the reflection of the globin chain unbalance only nor of the soluble pool of alpha hemoglobin chain but mainly is a reflection of insoluble alpha chains; and 3) the rate of proteolysis and instability of alpha chains are important factors which must be taken into consideration in the pathophysiology and the clinical heterogeneity of the disease.     

Synthesis of heterotrimeric collagen peptides containing the alpha1beta1 integrin recognition site of collagen type IV.

Collagen type IV provides a biomechanically stable scaffold into which the other constituents of basement membranes are incorporated, but it also plays an important role in cell adhesion. This occurs with collagen type IV mainly via the alpha1beta1 integrin, and the proposed epitope involved in this type of collagen/integrin interaction corresponds to a non-sequential R/Xaa/D motif, where the arginine and aspartate residues are provided by the alpha2 and alpha1 chains of the collagen molecule, respectively. Since the stagger of the three alpha chains in native collagen type IV is still unknown and different alignments of the chains lead to different spatial epitopes, two heterotrimeric collagen peptides containing the natural 457-469 sequences of the cell adhesion site were synthesized in which the single chains were assembled via disulfide bonds into the two most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. The differentiated triple-helical stabilities of the two heterotrimers suggest a significant structural role of the chain register in collagen, although the binding to alpha1beta1 integrin is apparently less affected as indicated by preliminary experiments.     

Purification and partial amino acid sequence of papain-solubilized class II transplantation antigens

Papain-solubilized human class II (HLA-DR) antigens have been purified from cadaveric spleens by ion-exchange chromatography, gel chromatography, and immunosorbent purification. The isolated papain-solubilized antigens comprised two subunits with apparent molecular weights of 23 000 and 30 000, respectively. The circular dichroism spectrum for the isolated class II antigens was similar to spectra recorded for HLA-A, -B, and -C antigens, immunoglobulins, and immunoglobulin fragments. Thus, class II antigens contain a considerable amount of beta structure. The small subunit (beta chain) exhibited extensive charge heterogeneity on two-dimensional isoelectric focusing polyacrylamide gel electrophoresis, whereas the large subunit (alpha chain) was more homogeneous. The structural heterogeneity of beta chains remained after neuraminidase treatment. The NH2-terminal amino acid sequence of the beta chains displayed multiple residues in several positions in accordance with the genetic polymorphism displayed by this chain. The alpha chain also displayed multiple residues in some positions, suggesting either that some of the genetic polymorphism of the class II antigens may be endowed in this chain or that multiple loci control the expression of several alpha chains. Papain-solubilized class II antigen subunits were homologous in their amino acid sequences with HLA-DR antigens of defined antigenic specificity as well as with murine I-E/C antigens.     

Synthetic heterotrimeric collagen peptides as mimics of cell adhesion sites of the basement membrane

Collagen type IV forms a network in the basement membrane into which other constituents of the tissue are incorporated. It also provides cell-adhesion sites that are specifically recognized by cell-surface receptors, i.e., the integrins. Different from the ubiquitous sequential RGD adhesion motif found in most of the matrix proteins, in collagen type IV, the responsible binding sites for alpha1beta1 integrin have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. Because of the heterotrimeric character of this collagen, the spatial geometry of the binding epitope depends not only on the triple-helical fold, but decisively even on the stagger of the chains. To investigate the effects of chain registration on the conformational properties and binding affinities of this adhesion epitope, two synthetic heterotrimeric collagen peptides consisting of the identical three chains were assembled by an artificial cystine knot in two different registers, i.e., in the most plausible alpha2alpha1alpha1' and less probable alpha1alpha2alpha1' chain alignment. A detailed conformational characterization of both trimers allowed to correlate their different binding affinities for alpha1beta1 integrin with the degree of local plasticity of the two different triple helices. Optimal local breathing of the rod-shaped collagens is apparently crucial for selective recognition by proteins interacting with these main components of the extracellular matrix.     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

Biosynthesis of the O-glycosidically linked oligosaccharide chains of fetuin. Indications for an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase with a narrow acceptor specificity in fetal calf liver

Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.     

Subunit inequivalence in superoxide anion formation during photooxidation of human oxyhemoglobin.

Subunit inequivalence in the photooxidation of human oxyhemoglobin was investigated by quantitative analysis of the fraction of alpha and beta chains oxidized after low intensity flash photolysis using white light in quartz cuvettes. This reaction was previously shown to generate methemoglobin and superoxide anion as photoproducts (Demma, L.S., and Salhany, J.M. (1977) J. Biol. Chem. 252, 1226-1230). The present results indicate that superoxide anion photodissociates from the alpha chain about 3 to 4 times more extensively than from beta. This difference was observed in the presence of superoxide dismutase and catalase at both pH 7 and 9, suggesting that photolysis is directly responsible. The reaction of superoxide anion with oxyhemoglobin was also studied with the same analytical methods and no chain differences could be observed. If the electronic structure of the oxyheme complex is viewed as a spin equilibrium between a singlet ground state and a charge transfer configuration, our results may indicate that the uv component of white light perturbs this equilibrium to a greater extent in the oxygenated alpha chain, implying that the separation of energy levels may be smaller in that chain as compared with beta.     

A new mode for heme-heme interactions in hemoglobin associated with distal perturbations.

The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.