Role of β112 cys (G14) in homo- (β4) and hetero- (α2β2) tetramer hemoglobin formation

In order to assess the role of beta 112 Cys in homo-and hetero-tetrameric hemoglobin formation, we expressed four beta 112 variants (beta(112Cys-->Asp), beta(112Cys-->Ser), beta(112Cys-->Thr) and beta(112Cys-->Val)) and studied assembly with alpha chains in vitro, beta 112 Cys is normally present at beta(1) beta(2), and alpha(1) beta(1) interaction sites in homo-(beta(4)) and hetero-tetramers (alpha(2) beta(2)). beta(4) formation in vitro was influenced by the amino acid at beta 112, beta 112 Asp completely inhibited formation of homo-tetramers, whereas beta 112 Ser showed only slight inhibition. In contrast, beta 112 Thr or Val enhanced homotetramer formation compared with PA chains. Association constants for homo-tetramer formation increased in the order of beta(112Cys-->Ser), beta(A), beta(112Cys-->Thr), and beta(112Cys-->Val), whereas the value for beta(112Cys-->Asp) was Zero under the same conditions. These beta 112 changes also affected in vitro alpha(2) beta(2), hetero-tetramer formation. Order of alpha(2) beta(2) formation under limiting alpha-globin chain conditions showed Hb beta C112S > Hb A > Hb S = Hb beta C112T = Hb beta C112V * Hb beta C112D, Hb beta 112D can form tetrameric hemoglobin, but this beta 112 change promotes dissociation into alpha and beta chains instead of cup dimer formation upon dilution, These results indicate that amino acids at beta(1) alpha(1) interaction sites such as beta 112 on the G; helix play a key role in stable ap dimer formation. Our findings suggest, in addition to electrostatic interaction between alpha and beta chains, that dissociation of beta(4) homo-tetramers to monomers and hydrophobic interactions of the beta 112 amino acid with alpha chains governs stable alpha(1) beta(1) interactions, which then results in formation of functional hemoglobin tetramers, Information gained from these studies should increase our understanding of the mechanism of assembly of multi-subunit proteins.