The rates of alpha/beta monomer combination of four beta (A) variants (beta 112C --> S, beta 112C --> D, beta 112C --> T, and beta 112C --> V) in the presence and absence of beta 16G --> D (beta (J)) were measured in an attempt to assess the consequences of amino acid substitution at both a surface (beta 16) and an alpha (1)beta (1) interface (beta 112) residue on oxyhemoglobin assembly. Rates of alpha/beta monomer combination determined spectrally in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM EDTA, pH 7.4, at 21.5 degreesC differed by over 40-fold (22 +/- 2.0 to 0.49 +/- 0.1 X 10(5) M-1 s(-1)), and were in the order: HbA beta 112S = HbJ beta 16D, beta 112S > HbA beta 112D = HbJ beta 16D, beta 112D > HbA > Hb J > HbA beta 112T = HbJ beta 16D, beta 112T > HbJ beta 16D, beta 112V > HbA beta 112V. This extensive kinetic investigation of single/double amino acid-substituted recombinant hemoglobin molecules, in conjunction with molecular modeling studies, has allowed examination of an array of unique alpha/beta subunit interactions and assembly processes.