Assembly of the gigantic hemoglobin of the earthworm Lumbricus terrestris. Roles of subunit equilibria, non-globin linker chains, and valence of the heme iron.

The extracellular hemoglobin of the earthworm Lumbricus terrestris has four major kinds of O2-binding chains: a, b, and c (forming a disulfide-linked trimer), and chain d. Non-heme, non-globin structural chains, "linkers," are also present. Light-scattering techniques have been used to show that the ferrous CO-saturated abc trimer and chain d form an (abcd)4 complex of 285 kDa at neutral pH. Formation of the full-sized 4-MDa molecule requires the addition of linker chains in the proportion of two linkers per (abcd)4 and occurs much more rapidly in the presence of 10 mM calcium. This stoichiometry is supported not only by direct quantitative analysis of the intact hemoglobin but also by the fact that the addition of 50% of the proposed stoichiometric quantity of linkers results in the conversion of 50% of the (abcd)4 to full-sized molecules. Isolated CO-saturated abc trimers self-associate to (abc)2 and higher aggregates up to an apparent limit of (abc)10 approximately 550 kDa. The CO-saturated chain d forms dimers, (d)2, and tetramers, (d)4. Oxidation of the (abcd)4 complex with ferricyanide causes complete dissociation of chain d from the abc trimer, but addition of CN- maintains the (abcd)4 complex. Valence hybrids have also been studied. The ferrous CO-saturated abc trimer and met (ferric) chain d also associate to form (abcd)4, but the met abc trimer and ferrous CO-saturated chain d do not. Oxidation of the abc trimer and chain d to the ferric form causes the formation of a characteristic hemichrome spectrum with a maximum at 565 nm and a shoulder near 530 nm. These results show that interactions between the abc trimer and chain d are strongly dependent on the ligand and valence state of the heme iron. Light-scattering measurements reveal that oxidation of the intact Hb produces a significant drop in molecular mass from 4.1 to 3.6 MDa. Inclusion of CN- prevents this drop. These experiments indicate that oxidation causes the Hb to shed subunits. The observations provide an explanation for the wide variations in the molecular mass of L. terrestris Hb that have been observed previously.

The extracellular hemoglobin of the earthworm, Lumbricus terrestris. Determination of subunit stoichiometry.

The giant extracellular hemoglobin of the earthworm, Lumbricus terrestris, has four major O2-binding chains, a, b, and c (forming a disulfide-linked trimer) and d ("monomer"). Participation of additional "linker" chains L1, L2, and L3 is necessary for the assembly of the approximately 3,900+ kDa two-tiered hexagonal structure. We have determined the proportions of linker chains, trimer, and chain d in the hemoglobin by reverse phase high performance liquid chromatography which resolves all of the components and also permits simultaneous determination of the heme content. The proportions of components were determined by two independent procedures: integration of the absorbance peaks at 220 nm and amino acid analysis of the peak fractions. The results indicate that the weight proportion of linker chains is 0.163 +/- 0.023. This value, together with molecular masses determined both by amino acid sequence analysis and by matrix-assisted laser desorption mass spectrometry, gives a molar ratio of abcd chains to linkers of 8:1, corresponding to the minimal unit (abcd)2.L. This ratio suggests that 24 (abcd)2 units and 24 linker chains form the complete structure with a total calculated mass of polypeptide of 3,975 kDa with hemes on chains a, b, c and d and on one linker. The calculated heme content is 3.1% not including carbohydrate. This accounts for a measured heme content of 3.0% on a polypeptide basis. Additional mass (approximately 133 kDa, 3.4%), attributed to carbohydrate, brings the total mass to 4,108 kDa with a minimum molecular mass/heme of 20,500 Da. The presence of equimolar quantities of three unique linker chains means that the apparent one-twelfth structural units seen by electron microscopy cannot all be identical.

Oxygen binding constants and stepwise enthalpies for human and bovine hemoglobin at pH 7.6.

A high-precision thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand-binding macromolecules in a manner analogous to that of the Gill thin-layer optical apparatus [Doleman & Gill (1976) Anal. Biochem. 87, 127]. We have generated differential heat-binding curves of oxygen binding to human and bovine hemoglobin in phosphate buffer at pH 7.6, with the enzyme-reducing system of Hayashi et al. [(1973) Biochim. Biophys. Acta 310, 309]. Experiments were conducted at a number of different temperatures in order to expand the data field, allowing for separation of enthalpy and free energy parameters. This type of experimental analysis makes no assumptions of optical linearity between the various heme groups and reveals that the triply ligated species is measurably significant for both human and bovine hemoglobin. It was also determined that the concentration of doubly ligated species of bovine hemoglobin is relatively low. The experiments indicate that the reactions for both hemoglobins are enthalpy-driven for oxygen stepwise additions 1, 2, and 4 while being entropy-driven for step 3. Human hemoglobin oxygen-binding experiments were also performed with the Gill thin-layer optical apparatus under solution conditions identical to those used in the calorimeter. The experiments revealed that if optical linearity is assumed, the overall third equilibrium constant is negative or near zero. This indicated that either the optical cell's performance is much poorer than the thin-layer calorimeter or there is an appreciable nonlinear optical effect.

Nonlinear optical effects in oxygen-binding reactions of hemoglobin A0.

Optical spectra have been taken in the Soret band (440-400 nm) under different oxygen partial pressures for hemoglobin (Hb) A0 at pH 7.0, 15 degrees C, 2-3 mM heme, 30 mM inositol hexaphosphate, 0.1 Hepes and 0.1 M NaCl. Application of the matrix method of singular value decomposition (SVD) to the difference spectra for different oxygen pressures shows the presence of at least two distinct optical transitions. From this result one concludes that the optical response to oxygen binding is nonlinear in the Soret band. The degree of nonlinearity has been determined by fitting the data at different wavelengths to the four-step reaction Adair equation with the inclusion of optical parameters that describe the intermediate oxygenated species. It is found that the data are well-represented by two optical parameters at each wavelengths, one which represents the optical change for the addition of the first and second oxygen molecules and the other which corresponds to the change for the addition of the third and fourth oxygen molecules. The ratio of these optical parameters depends only moderately upon wavelength with an average value of 0.8 over the Soret band. Thus, there is an approx. 20% smaller optical response for the first two ligated species than that for the last two ligated species. The overall Adair equilibrium constants are evaluated as follows: beta 1 = 0.081 +/- 0.003 Torr-1, beta 2 = 2.53 x 10(-3) +/- 2.4 x 10(-4) Torr-2, beta 3 = 1.25 x 10(-5) +/- 1.0 x 10(-6) Torr-3, beta 4 = 1.77 x 10(-6) +/- 1.5 x 10(-7) Torr-4.