Bovine Hemoglobin Cross-Linked through the β Chains

Abstract

2-Nor-2-formylpyridoxal (NFPLP) has been synthesized and coupled to bovine Hb according to the procedure developed by Benesch and Benesch. The reaction of bovine Hb with NFPLP leads to a cross-linkage between the beta subunits, which greatly stabilizes the low affinity T state of the molecule and simultaneously abolishes the tendency of the tetramer to dissociate into alpha beta dimers. The functional properties, examined from both the equilibrium and kinetic points of view, indicate that the chemical modification affects the O2 affinity, abolishes cooperativity, and induces a slight decrease of the Bohr effect. From modeling studies we are confronted with two different structural alternatives; the cross-link of beta chains may be formed between lysine 82 of beta2 and the N terminus of methionine 2 of beta1 or between the two lysine 82 residues of both beta2 chains. Digestion of modified beta globin chains and isolation of the cross-linked peptide have showed that NFPLP cross-links Met-beta2 and Lys-beta82. This allowed discussion in some detail of the molecular basis of the Bohr effect of the modified bovine hemoglobin. On the whole, NFPLP-modified bovine Hb could be considered as a first step toward the synthesis of a potential blood substitute.

POLAR BEAR HEMOGLOBIN AND HUMAN Hb A0: SAME 2,3-DIPHOSPHOGLYCERATE BINDING SITE BUT ASYMMETRY OF THE BINDING?

“…Samples were stripped of organic phosphates by passage through a mixed bed resin as previously described, [8] using a Sephadex G-25 column (2-4 C) equilibrated with 10 mM Tris-HCl, containing 0.1 M NaCl. The concentration in aliquots was adjusted to 0.3 g=dL, and 0.025 mL was subjected to electrophoresis for 30 mins at 350 volts at 4 C using Tris-EDTA-glycine buffer.…”

Anionic Binding Site and 2,3-DPG Effect in Bovine Hemoglobin

“…Chloride and any organic phosphates were removed by gel filtration on a Sephadex G-25 column (at 2-4 °C) and equilibrated with 10 mM Tris-HCl (pH 8.0), containing 0.1 M NaCl. Subsequently, the product was passed through a column of a mixed-bed ion-exchange resin (Bio-Rad AG 501-X8) (9). Spectrophotometric Measurements.…”

Spectroscopic study of gamma irradiated bovine hemoglobin

“…This means the deterioration of tyrosine molecular consistence, which confirms EPR spectroscopic analysis. Moreover, most of other bands showed decrease in intensity: marked decrease in the amide I band (about 5.78%) upon irradiation (743 Gy of Cs-137 gamma radiation) may be attributed to a change in a-helix or in the parallel or the anti-parallel b-sheets (Marta et al, 1996).…”

“…It is composed of two pairs of non-identical subunits, alpha and beta. Each alpha-beta pair is more closely associated than they are with each other, but the overall arrangement is roughly tetrahedral (http://www.bmb.uga.edu/ wampler/tutorial/prot4.html) (Marta et al, 1996).…”