Contact inhibition within hemoglobin S polymer by thiol reagents.
N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted beta 93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + HbS and HbA + HbS NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid alpha 2A beta A beta S, alpha 2A beta A,NEM beta S or alpha 2A beta A beta S,NEM respectively and 25% parental molecules as confirmed by ion-exchange HPLC performed in anaerobic conditions. Within the hybrid molecule, beta A or beta A,NEM chain has to be present in the alpha beta dimer located in trans to the dimer which contains the only beta 6 valine residue participating in intermolecular contacts (dimer in cis), while beta S or beta S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + HbS and 20% for HbA + HbS NEM mixtures compared to HbA + HbS mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of HbS + Hb Créteil beta 89 Ser----Asn. The beta 89 residue in trans next to the cysteine beta 93 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the alpha chains.
D002498:Centrifugation; D003460:Crystallization; D005033:Ethylmaleimide / Q000494:pharmacology; D006441:Hemoglobin A / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000378:metabolism*; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism*; D011487:Protein Conformation; D012995:Solubility; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D006451:Hemoglobin, Sickle; D011108:Polymers; D013439:Sulfhydryl Reagents; D006441:Hemoglobin A; D005033:Ethylmaleimide; D007460:Iodoacetamide