Subunit structure and dissociation of Callinectes sapidus hemocyanin.

The hemocyanin of the blue crab, Callinectes sapidus has two major components sedimenting with approximate sedimentation coefficients of 17 S and 25 S. Molecular weight data based on light scattering and sedimentation equilibrium measurements at pH 7.8 suggest that the two components have molecular weights of approximately 450 000 and 900 000 in the presence of stabilizing Ca2+. In the absence of Ca2+, the molecular weights are found to be about 5% lower, suggesting some dissociation of the hemocyanin components. Circular dichroism and optical rotatory dispersion measurements in the far-ultraviolet region gave nearly identical spectra for the two components. Based on the reference parameters of Chen et al. (Chen, Y.H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131 and Chen, Y.H., Yang, J.T. and Chan, K.H. (1974) Biochemistry 13, 3340--3359), estimates of 16--20% alpha-helix, 40--60% beta-structure, and 30--40% random organization were obtained for the two hemocyanin components. Exposure to 6 M Gdn HCl gave light scattering molecular weights of approx. 68 000 and 77 000, which is close to one-sixth of the molecular weight of the 17 S component. These results support the view that the two components of C. sapidus hemocyanin share the hexameric and dodecameric organization common to arthropod hemocyanins. The salts of the Hofmeister series and the ureas are found to dissociate the dodecameric component with the former exhibiting the usual order of effectiveness of NaCl, NaBr, NaI, and NaClO4 dissociation, while the ureas show an inverse order of decreasing effectiveness in going from urea to methyl-, ethyl- and propylurea. This suggests that polar and ionic interactions are relatively more important than hydrophobic interactions for the stabilization of the dodecameric form of C. sapidus hemocyanin. The dissociation behavior of the 17 S hexameric species by GdnHCl in the 0--1.5 M concentration region (where essentially no denaturation occurs), based on light scattering molecular-weight measurements, is satisfactorily accounted for by equations describing the dissociation of hexamers to monomers.

On the mechanism for the red-cell accumulation of mefloquine, an antimalarial drug.

The accumulation of the antimalarial drug mefloquine by human red blood cells has been studied by 19F-NMR spectroscopy. The uptake process was nonlinearly dependent on the external drug concentration. Concentrations inside cells as high as 60-times greater than those in the extracellular phosphate-buffered-saline were observed. Red-cell ghosts were also found to accumulate mefloquine with high-affinity binding sites for the drug. Hemoglobin was found to bind mefloquine with low affinity, but due to the high concentration of this protein it is a significant drug compartment in the red cell. Analysis of the 19F-NMR chemical shifts and linewidths of mefloquine in the presence of red cells, red-cells ghosts and hemoglobin indicates restricted mobility of the drug in the membrane-bound state and slow exchange with the extracellular medium. This is a significant characteristic of the reaction in connection with the prophylactic activity of the drug. Exchange of the drug between hemoglobin and the red-cell membrane, however, is fast and may play an important role in the bioavailability of the drug to the parasite.

Reaction of acetaldehyde with hemoglobin.

Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.

Dimeric hemoglobins from the arcid blood clam, Noetia ponderosa. Structure and functional properties.

The hemoglobin found in the nucleated erythrocytes of the arcid blood clam Noetia ponderosa is heterogeneous and consists of two electrophoretic components, Hb-Major and Hb-Minor, present in about 80% and 20% proportions, respectively. Both components are hemoglobin dimers over a wide concentration range based on light-scattering measurements. No higher aggregation states are observed. The oxygen binding by Hb-Major and Hb-Minor is characterized by p50 values of 16.8 and 8.7 mm of Hg and Hill coefficients of 1.4 and 1.2, respectively, at pH 7.0 and 25 degrees C. Neither component exhibits an alkaline Bohr effect. An unusual nonlinear Hill plot is observed for Hb-Major. Hb-Major is composed of two different polypeptide chains and thus is a heterodimer based on sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography. By the same methods, Hb-Minor is a homodimer and may share a common chain with Hb-Major. Amino acid compositions of the two hemoglobins indicate 2 histidines/polypeptide chain which are presumably involved in the coordination of the heme iron. Visible absorption spectra indicate the heme environment is normal in the oxy state but perhaps more constrained in the deoxy state. Oxygen binding as a function of temperature and concentration and binding by the intact erythrocytes indicates the absence of intracellular regulators of oxygen binding.

Reaction of tobacco smoke aldehydes with human hemoglobin.

Formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, and acrolein, all of which are constituents of tobacco smoke, were reacted in 5 mM concentration with the purified major fraction of normal adult human hemoglobin (hemoglobin Ao) in 1 mM concentration. A cigarette smoke condensate, diluted to contain 5 mM total aldehydes, was also reacted with 1 mM hemoglobin Ao. Cationic exchange high-performance liquid chromatography (HPLC) showed that the products formed from simple aliphatic aldehydes, with the exception of formaldehyde, were analogues of those formed from acetaldehyde, earlier shown by us to be imidazolidinone derivatives, that is, cyclic addition products of the N-terminal aminoamide function of alpha and beta chains. Formaldehyde and acrolein produced a heterogeneous mixture of derivatives including cross-linked hemoglobin dimers. The greater proportion of modified hemoglobins produced by condensate aldehydes resembled those formed from acetaldehyde, the most abundant aldehyde in the condensate. A smaller fraction consisted of cross-linked hemoglobin dimers, presumably due to the action of formaldehyde. Mass spectrometric and HPLC analyses of the 2,4-dinitrophenylhydrazones precipitated from the condensate documented the presence of formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, furfural, and methylfurfural. The toxicity of aldehydes is briefly discussed in the context of the findings of this study.

Light-scattering investigations of the subunit dissociation of human hemoglobin A. Effects of various neutral salts.

The effectiveness of various salts of the Hofmeister series as dissociating agents for human hemoglobin A tetramers has been investigated by light-scattering molecular-weight measurements. Dissociation of hemoglobin to half-molecules of alpha beta dimers follows the order of the series dictated predominantly by the sequence of the anions F- less than Cl- less than Br- less than ClO4- less than SCN-, I-, with the cations Na+ and K+ having relatively little effect on the observed dissociation. The use of equations derived for predicting the effects of dissociating reagents on the structure of subunit proteins [Herskovits, T. T., and Ibanez, V. S. (1976), Biochemistry 15, 5715] together with Setschenow constants based on the model amino acid data of Nandi and Robinson were found to give a satisfactory account of the dissociation behavior observed with many of the salts, giving reasonable estimates of the number of amino acids that form the smaller contact area of the alpha beta subunits of hemoglobin shown by the Perutz crystallographic model. The analysis of the dissociation data also extends the utility of the Setschenow constants tested for the characterization of the dissociation behavior of other subunit proteins.

Intrinsic fluorometric determination of the stable state of aggregation in hemoglobins.

Front-face fluorometry can detect steady-state intrinsic fluorescence of hemoglobins (R. E. Hirsch, R. S. Zukin, and R. L. Nagel, 1980, Biochem. Biophys. Res. Commun. 93, 432-439), a property that can be used to study the dimerization of human hemoglobins (R. E. Hirsch, N. A. Squires, C. Discepola, and R. L. Nagel, 1983, Biochem. Biophys. Res. Commun. 116, 712-718). We report that the stable dimeric hemoglobin components of the arcid clams Noetia ponderosa and Anadara ovalis exhibit fluorescence emission maxima shifted to longer wavelengths compared to tetrameric human hemoglobin. Conversely, the tetrameric major hemoglobin (Hb) component of A. ovalis exhibits an emission maximum similar to that of tetrameric Hb A. Hence, stable dimeric hemoglobins can be detected by emission maxima at longer wavelengths relative to Hb A. Fluorescence studies of ligand binding to these clam hemoglobins indicate structural and functional differences among these components and compared to Hb A. We conclude that different stable aggregation states of hemoglobins may be determined by intrinsic fluorescence when studied with front-face optics.

Light-scattering investigation of the subunit dissociation of Homarus americanus hemocyanin. Effects of salts and ureas.

The subunit dissociation of the hemocyanin of the lobster, Homarus americanus, by the various salts of the Hofmeister series and the hydrophobic reagents of the urea-guanidinium chloride (GdmCl) class was investigated by laser light scattering molecular weight measurements. The dissociations of the hemocyanin dodecamers to hexamers by the various salts and the lower members of the urea series are found to be rapid and reversible, as predicted by the mass action law for monomer-dimer type of reactions. The salts are found to be very effective dissociating reagents with the unusual order of increasing effectiveness, Cl- less than Br- less than I- less than ClO4-, SCN-. The ureas and GdmCl are found to be relatively ineffective dissociating agents. In addition, the ureas show a decreasing order of effectiveness in going from urea to methyl-, ethyl-, and propylurea. This suggests that hydrophobic interactions are not the dominant stabilizing forces between the pairs of hexamers that form the dodecameric structure. Polar and ionic interactions appear to be the major stabilizing forces of the dodecameric structure. The use of equations derived for predicting the effects of dissociating reagents and salts on the structure of subunit proteins [Herskovits, T. T., & Ibanez, V. S. (1976) Biochemistry 15, 5715-5721] together with binding and Setschenow constants based on model amino acid data is found to give good account of the dissociation behavior observed with the salts, urea, and methylurea in the presence of calcium ion at both pH 7.8 and pH 9.5. The apparent number of amino acids at the contact areas of the hexamers, Napp, required to fit the dissociation data were found to be 24 +/- 8 at pH 7.8 and 23 +/- 4 at pH 9.5. However, because of the possible effects of molecular microheterogeneity, the estimates of amino acids at the contact areas must be viewed with caution, depending on further investigations.

Anadara ovalis hemoglobins: distinct dissociation and ligand binding characteristics.

The red cells of the arcid clam Anadara ovalis contain two electrophoretically distinct hemoglobins: Hb Major (Hb Ma) and Hb Minor (Hb Mi). The major component consists of two electrophoretically indistinguishable tetramers each composed of two heterodimers; the minor hemoblogin is a homodimer whose subunits are different from the tetramer. Functionally, Hb Ma has a higher P50, exhibits a concentration dependent oxygen affinity, has significant ligand cooperativity (n = 2.0), lacks a Bohr effect and is unaffected by ATP. HB Mi has a P50 which is lower and independent of hemoglobin concentration, shows appreciable cooperativity (n = 1.4) and exhibits no heterotropic effects. Both Hb Ma and Mi are resistant to dissociation in the presence of 1.0 M NaI, NaCl and guanidine-HCl but dissociate to monomers when converted to the aquamet but not the cyanmet derivative. The dissociation is completely inhibited by mercaptoethanol. The large number of reactive -SH groups (10-13 per tetramer) suggests that the monomerization is mediated by intra-subunit disulfide bridge formation.