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

摘要

The hexagonal bilayer hemoglobin (Hb) of the leech Macrobdella decora has an equilibrium sedimentation mass of 3544(+/- 80) kDa. Maximum entropy analysis of the electrospray ionization mass spectra of the Hb show three groups of peaks: two peaks of equal intensity at similar to 17 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 relative intensities of 1:6:3, respectively; and three peaks of equal intensities 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 the 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 Da), R (16,666.7 Da) and S (16,792.9 Da), indicating that F, G and H represent disulfide-bonded dimers of globin chains, P + Q, P + R and P + S, respectively. The relative intensities of the three groups of peaks 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 with 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 10(5) 1(3) g(-3). A model of the Hb consisting of a hexagonal bilayer of 36 tetramer and 42 linker subunits provides a total mass and globin to linker ratio closest to the experimental values. Equilibrium O-2 binding measurements of the native Hb and its tetramer and monomer subunits 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 exhibits a moderately high Oz affinity, P-50 = 4.4 torr at PH 7.5 and 25 degrees C, a high cooperativity (n(50) similar to 3) and a substantial Bohr effect, phi = Delta logP,(50)/Delta pH = -0.38. The tetramer subunit 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 has a much higher affinity (P-50 = 0.29 torr) and no cooperativity or Bohr effect. Analysis of the O-2 binding curves in terms of the two-state (MWC) allosteric model showed a markedly increased K-T in the tetramers compared with the native Hb, resulting in opposite relative Delta G values. Increases in cation (Ca2+, Mg2+) concentrations and pH increase 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. The results suggest that the overall cooperativity of O-2 binding of the Hb consists of the cooperativity of the tetramer subunit and an additional contribution due to the hexagonal bilayer structure, requiring globin and linker chains. (C) 1995 Academic Press Limited [References: 66]