Characterization of the binding of the anti-sickling compound, BW12C, to haemoglobin

Abstract

The anti-sickling agent BW12C [Beddell, Goodford, Kneen, White, Wilkinson & Wootton (1948) Br. J. Pharmacol. 82, 397-407] was designed to left-shift the oxygen saturation curve of haemoglobin (HbA) by preferential binding to the oxy conformation at a single site between the terminal amino groups of the α-chains through Schiff's base formation, ionic and hydrophobic interactions. In the present work, Schiff's base linkages formed with [14C]BW12C were reduced with NaBH4 and the α- and β-globin chains separated. Under oxy conditions at a molar ratio of 2:1, the covalently bound BW12C is localized almost exclusively on a single α-chain; tryptic digestion confirms the terminal amino group (α1-valine) as the reaction site, in accord with the design hypothesis. However, about half the labelled BW12C is released on tetramer disruption, suggesting the presence of additional non-covalent binding. Under deoxy conditions, α- and β-chains are labelled approximately equally, and at higher molar ratios additional binding in both oxy and deoxy conditions is seen. Isoelectric-focusing studies under oxy conditions show a complex pattern of modified bands for both HbA and HbA(1c) (blocked β-terminal amino groups) but no modification for HbA carbamylated at both α- and β-terminal amino groups or at the α-chains only, again confirming the α-terminal amino region as the main interaction site. Equilibrium dialysis measurements under oxy conditions indicate two strong binding sites with a binding constant of less than 10-6 M and a number of weaker binding sites. The present data thus confirm that BW12C binds at the intended locus but reveal additional non-covalent binding at an undefined site, and weaker binding through Schiff's base formation with other amino groups.