MASS-SPECTROMETRIC COMPOSITION, MOLECULAR-MASS AND OXYGEN-BINDING OF MACROBDELLA-DECORA HEMOGLOBIN AND ITS TETRAMER AND MONOMER SUBUNITS

The hexagonal bilayer hemoglobin (Hb) of the leech Macrobdella decora has an equilibrium sedimentation mass of 3544(+/- 80) kDa. Maximum ent ropy analysis of the electrospray ionization mass spectra of the Hb sh ow three groups of peaks: two peaks of equal intensity at similar to 1 7 kDa, A (16,770.1 Da) and B (16,841.9 Da); three peaks at similar to 24 kDa, C (24,340.1 Da), D (24,398.6 Da) and E (24,420.0 Da) with rela tive intensities of 1:6:3, respectively; and three peaks of equal inte nsities at similar to 33 kDa, F (32,586.0 Da), G (32,714.5 Da) and H ( 32,849.9 Da). Although reduction with dithiothreitol does not affect t he masses of peaks A through E, the similar to 33 kDa peaks give rise to four new peaks at similar to 16 kDa, P (16,052.2 Da), Q (16,537.3 D a), R (16,666.7 Da) and S (16,792.9 Da), indicating that F, G and H re present disulfide-bonded dimers of globin chains, P + Q, P + R and P S, respectively. The relative intensities of the three groups of peak s are (A + B) to (C + D + E) to (F + G + H) = 0.39:0.26:0.32, and the globin to linker ratio 0.71:0.29 is in good agreement with the ratio 0 .72:0.28 obtained by HPLC. The largest functional subunit obtained by dissociation at PH 7 in 4 M urea, is a subunit lacking linker chains w ith apparent mass 63(+/-3) kDa. The equilibrium sedimentation profile of this subunit is fitted best as a monomer-dimer-tetramer equilibrium , with association constants K-1,K-2=365 l g(-1) and K-1,K-4 = 8.1 x 1 0(5) 1(3) g(-3). A model of the Hb consisting of a hexagonal bilayer o f 36 tetramer and 42 linker subunits provides a total mass and globin to linker ratio closest to the experimental values. Equilibrium O-2 bi nding measurements of the native Hb and its tetramer and monomer subun its were carried out over the pH range 6.6 to 8.0 at 10 and 25 degrees C, and in the absence and presence of Na+, Mg2+ and Ca2+. The Hb exhi bits a moderately high Oz affinity, P-50 = 4.4 torr at PH 7.5 and 25 d egrees C, a high cooperativity (n(50) similar to 3) and a substantial Bohr effect, phi = Delta logP,(50)/Delta pH = -0.38. The tetramer subu nit has a higher affinity, lower cooperativity and smaller Bohr effect , 1.9 torr, 1.3 to 1.5 and -0.30, respectively. The monomer subunit ha s a much higher affinity (P-50 = 0.29 torr) and no cooperativity or Bo hr effect. Analysis of the O-2 binding curves in terms of the two-stat e (MWC) allosteric model showed a markedly increased K-T in the tetram ers compared with the native Hb, resulting in opposite relative Delta G values. Increases in cation (Ca2+, Mg2+) concentrations and pH incre ase O-2 binding affinity as well as K-R and K-T of the Hb but have no effect of the properties of either the tetramer or monomer subunits. T he results suggest that the overall cooperativity of O-2 binding of th e Hb consists of the cooperativity of the tetramer subunit and an addi tional contribution due to the hexagonal bilayer structure, requiring globin and linker chains. (C) 1995 Academic Press Limited