Mass spectrometric composition, molecular mass and oxygen binding of Macrobdella decora hemoglobin and its tetramer and monomer subunits

Abstract

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 approximately 17 kDa, A (16,770.1 Da) and B (16,841.9 Da); three peaks at approximately 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 approximately 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 approximately 33 kDa peaks give rise to four new peaks at approximately 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 K1,2 = 365 l g-1 and K1,4 = 8.1 x 10(5) l3 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 O2 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 O2 affinity, P50 = 4.4 torr at pH 7.5 and 25 degrees C, a high cooperativity (n50 approximately 3) and a substantial Bohr effect, phi = delta log P50/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 (P50 = 0.29 torr) and no cooperativity or Bohr effect.(ABSTRACT TRUNCATED AT 400 WORDS)