Austen Riggs

[1] Preparation of blood hemoglobins of vertebrates

Publisher Summary This chapter deals with the preparation of blood hemoglobins of vertebrates. The hemoglobins of vertebrates are the most extensively studied of any proteins, largely because of their ease of preparation. The red cells are easily separated from the plasma by centrifugation. Hemolysis leads immediately to a solution that is usually more than 90% pure. Nevertheless, precautions need to be taken to obtain preparations of maximum value. Hemoglobins from different organisms vary greatly in their stability and resistance to denaturation, tendency to oxidize to methemoglobin, solubility, and chromatographic and electrophoretic properties. What is appropriate for mammalian hemoglobins is frequently inappropriate for those of lower vertebrates. The chapter explains erythrocyte preparation. Blood from small animals is usually best obtained by cardiac puncture. Large animals can be bled from superficial veins—for example, those of the ears of rabbits and elephants. The caudal vein of fish is an excellent location for bleeding; it is usually easy, with a little practice, to insert a needle just ventral to the lateral line directly into the vein. The extraction of hemoglobin from very small animals and embryos presents special problems. The chapter also describes the preparation of hemolysate.

Nitric-oxide Dioxygenase Activity and Function of Flavohemoglobins SENSITIVITY TO NITRIC OXIDE AND CARBON MONOXIDE INHIBITION

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae,Alcaligenes eutrophus, and Escherichia colishare similar spectra, O2, NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V max) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme−1 s−1, respectively, at 37 °C with 200 μm O2. The K M values for NO are low and range from 0.1 to 0.25 μm.V max/K M (NO) ratios of 900–2900 μm −1 s−1 indicate an extremely efficient dioxygenation mechanism. ApproximateK M values for O2 range from 60 to 90 μm. NO inhibits the dioxygenases at NO:O2ratios of ≥1:100 and makes true K M (O2) values difficult to determine. High and roughly equal second order rate constants for O2 and NO association with the reduced flavoHbs (17–50 μm −1 s−1) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O2(K I (CO) = ∼1 μm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O2 from inhibitory NO and CO.

Lobster Hemocyanin: Properties of the Minimum Functional Subunit and of Aggregates

Lobster hemocyanin dissociates into a functional subunit of 68,000 to 70,000 molecular weight when Ca2+ ions are removed from an alkaline solution of low ionic strength. Succinylation results in a further dissociation into two nonfunctional subunits of approximately 34,000 to 35,000 molecular weight. Amino acid analysis and tryptic peptide patterns indicate that the functional subunit is composed of at least two polypeptide chains which are similar.

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 ∼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.

Identification of Myoglobin in Human Smooth Muscle

Myoglobin (Mb) has been believed to be absent generally from mammalian smooth muscle tissue. Examination of human rectal, uterine, bladder, colon, small intestine, arterial, and venous smooth muscle by immunohistochemical techniques shows that each of these tissues is immunopositive for both smooth muscle myosin and human Mb. Mb-specific primers were used for the polymerase chain reaction to generate cDNA from smooth muscle tissues. Southern hybridization with a Mb-specific probe gave a very strong signal with the cDNA from rectum, weaker signals from small intestine and uterus, a faint signal from colon, and no signal from bladder tissue. High performance liquid chromatography analysis coupled with sequence determination has shown that contaminating heme-binding serum albumin as well as hemoglobin in extracts of smooth muscle seriously compromise any heme-based or spectrophotometric assay of Mb. Combined affinity and size exclusion chromatography, however, provide the necessary resolution. The cDNA-derived amino acid sequence of human smooth muscle Mb was found to be identical to that of Mb from striated muscle.

PROPERTIES OF HEMOCYANINS. I. THE EFFECT OF CALCIUM IONS ON THE OXYGEN EQUILIBRIUM OF CRAYFISH HEMOCYANIN.

Abstract 1. 1. The oxygen affinity of hemocyanin from the crayfish, Procambarus simulans , is greatly decreased by dialysis between pH 6·5 and 8·0. This effect is largely reversed by 10 mM calcium or by 60 mM magnesium. 2. 2. The data do not conform to Hills equation, since the value of n increases with degree of oxygenation and is a function both of the calcium concentration and of pH. At low degrees of oxygenation n ≅1·5 and is independent of pH or calcium, while at high oxygenation levels n increases with either pH or calcium. 3. 3. The importance of calcium in controlling the degree of dissociation of hemocyanin into subunits is known. Since the calcium concentration in crayfish blood changes during the molting cycle, it seems possible that calcium may play a role in modifying the oxygen transport function of hemocyanin by changing the degree of aggregation.

Polymerization of hemoglobins of mouse and man. Structural basis.

Human hemoglobin P�rto Alegre and mouse hemoglobin (BALB/cJ) polymerize by forming intermolecular disulfide bridges. Both hemoglobins have externally oriented, reactive cysteinyl residues in the A-helix of the beta chain. Hemoglobin P�rto Alegre can be designated as α2 β29 Ser → Cys. The crysteinyl residue of the mouse hemoglobin is at position 13 in the beta chain.

Hemoglobin Polymerization in Mice

Polymerization of certain mouse hemoglobins to eight-chain double molecules is completely inhibited by iodoacetamide. Each double molecule appears to consist of two α2β2-units linked by way of their β-chains with two disulfide bridges.

The 109 Residue Nerve Tissue Minihemoglobin from Cerebratulus lacteus Highlights Striking Structural Plasticity of the α-Helical Globin Fold

A very short hemoglobin (CerHb; 109 amino acids) binds O(2) cooperatively in the nerve tissue of the nemertean worm Cerebratulus lacteus to sustain neural activity during anoxia. Sequence analysis suggests that CerHb tertiary structure may be unique among the known globin fold evolutionary variants. The X-ray structure of oxygenated CerHb (R factor 15.3%, at 1.5 A resolution) displays deletion of the globin N-terminal A helix, an extended GH region, a very short H helix, and heme solvent shielding based on specific aromatic residues. The heme-bound O(2) is stabilized by hydrogen bonds to the distal TyrB10-GlnE7 pair. Ligand access to heme may take place through a wide protein matrix tunnel connecting the distal site to a surface cleft located between the E and H helices.

The Structural and Functional Analysis of the Hemoglobin D Component from Chicken

Oxygen binding by chicken blood shows enhanced cooperativity at high levels of oxygen saturation. This implies that deoxy hemoglobin tetramers self-associate. The crystal structure of an R-state form of chicken hemoglobin D has been solved to 2.3-Å resolution using molecular replacement phases derived from human oxyhemoglobin. The model consists of an α2β2 tetramer in the asymmetric unit and has been refined to a R-factor of 0.222 (R-free = 0.257) for 29,702 reflections between 10.0- and 2.3-Å resolution. Chicken Hb D differs most from human oxyhemoglobin in the AB and GH corners of the α subunits and the EF corner of the β subunits. Reanalysis of published oxygen binding data for chicken Hbs shows that both chicken Hb A and Hb D possess enhanced cooperativity in vitro when inositol hexaphosphate is present. The electrostatic surface potential for a calculated model of chicken deoxy-Hb D tetramers shows a pronounced hydrophobic patch that involves parts of the D and E helices of the β subunits. This hydrophobic patch is a promising candidate for a tetramer-tetramer interface that could regulate oxygen binding via the distal histidine.