Oxygen-binding Proteins Research Articles

Hemocyanin respiratory pigment in bivalve mollusks.

Hemocyanins, high molecular weight oxygen-binding proteins, were identified in two species of protobranch bivalve mollusks, Acila castrensis and Yoldia limatula. Although hemocyanins have been reported in chitons, gastropods, and cephalopods, they have not been observed in the Class Bivalvia. In A. castrensis the dissociation products of hemocyanin, characterized by gel electrophoresis, had a subunit molecular weight of approximately 250K. Negatively stained preparations of extracted hemocyanin formed protein aggregates in the shape of cylinders measuring 35 by 38 nanometers. X-ray microanalysis of hemocyanin aggregates in thin sections of Y. limatula demonstrated the presence of copper in the molecules. The discovery of hemocyanin in the protobranchs reinforces the primitive nature of the taxon and is further evidence that the major molluscan classes have a common ancestry.

The detection of leghemoglobin-line sequences in legumes and non-legumes

Leghemoglobin is a major component of the nitrogen-fixing nodules formed by legumes in association with bacterial symbionts of the genusRhizobium. It is thought to be involved in regulating the oxygen tension within nodules. In a series of Southern blot experiments using cloned soybean leghemoglobin cDNAs as hybridization probes, cross-hybridizing sequences have been detected in legumes closely related to soybean (members of the Leguminosae subfamily Papilionoideae), as well as in a distantly related legume not reported to be nodulated (subfamily Caesalpinioideae). With the same probes, the presence of cross-hybridizing sequences has also been detected in plants outside the Leguminosae, including two nitrogen-fixing non-legumes and one species which is not nodulated. These results suggest that the genes for oxygen-binding proteins may be more widely dispersed than previously thought.

Comparison of the molecular size and shape of the extracellular hemoglobins of Tubifex tubifex and Lumbricus terrestris

The extracellular hemoglobins of Tubifex tubifex and Lumbricus terrestris have been examined by gel filtration and scanning transmission electron microscopy and a detailed comparison of their morphologies has been performed using digital image processing techniques. The hemoglobins possess identical elution volumes on analytical columns of Sepharose CL-6B and CL-2B. In electron micrographs the overall shape of negatively stained Tubifex and Lumbricus hemoglobins is similar to the two-tiered hexagonal appearance of other annelid extracellular hemoglobins. Tubifex hemoglobin possesses a vertex to vertex diameter of 31.3 nm and a height of 21.1 nm, while the corresponding dimensions of Lumbricus hemoglobin are 31.2 nm and 21.5 nm, respectively. These dimensions are about 25% larger than those obtained by conventional transmission electron microscopy but are consistent with other values obtained by scanning transmission EM (Kapp, O.H., Vinogradov, S.N., Ohtsuki, M. and Crewe, A.V. (1982) Biochim. Biophys. Acta 704, 546–548) and by X-ray diffraction of wet crystals of Lumbricus hemoglobin (Royer, W.E., Broden B.C., Jacobs, H.C. and Loves, W.E. (1981) in Invertebrate Oxygen-Binding Proteins (Lamy, J. and Lamy, J., eds.), 337–341, Marcel Dekker, New York). Comparison of the morphologies of the two hemoglobins by examination of individual pixel lines through their digital projections suggests that the molecules are very similar in overall structure.

Mitochondrial function in the presence of myoglobin.

The effect of myoglobin on oxygen consumption and ATP production by isolated rat skeletal muscle mitochondria was studied under steady-state conditions of oxygen supply. A method is presented for the determination of steady-state oxygen consumption in the presence of oxygen-binding proteins. Oxygen consumed in suspensions of mitochondria was replenished continuously by transfer from a flowing gas phase. Liquid-phase oxygen pressure was measured with an oxygen electrode; the gas-phase oxygen concentration was held constant at a series of fixed values. Oxygen consumption was determined from the characteristic response time of the system and the difference in the steady-state gas- and liquid-phase oxygen concentrations. ATP production was determined from the generation of glucose 6-phosphate in the presence of hexokinase. During steady-state mitochondrial oxygen consumption, the oxygen pressure in the liquid phase is enhanced when myoglobin is present. Functional myoglobin present in the solution had no effect on the relation of mitochondrial respiration and ATP production to liquid-phase oxygen pressure. Myoglobin functions in this system to enhance the flux of oxygen into the myoglobin-containing phase. Myoglobin may function in a similar fashion in muscle by increasing oxygen flux into myocytes.

Invertebrate Oxygen-Binding Proteins: Structure, Active Site, and Function

Invertebrate Oxygen-Binding Proteins: Structure, Active Site, and Function

2] Preparation of myoglobins

This chapter presents procedures for the isolation of intracellular oxygen-binding proteins of tissues, called “tissue hemoglobins” in the widest sense. All of these, except Ascaris and yeast hemoglobin, are monomers or dimers having a minimum molecular weight of 18,000 with similar optical spectra and chemical reactivity. Strictly, only muscle hemoglobin should be called “myoglobin”; by extension the term is often applied to other tissue hemoglobins as well. Ferric myoglobin may be purified by chromatography on carboxymethyl (CM) cellulose, usually at slightly acid pH or on diethylaminoethyl (DEAE) cellulose. The choice of preparative procedure depends on the use to which the purified myoglobin will be put. Both DEAE and CM ion-exchange columns yield myoglobin that is pure in the sense of being free from contaminating polypeptide chains. Better resolution of forms of myoglobin differing only in charge is achieved on CM-cellulose. Such columns, however, are usually operated at acid pH, and it is a matter of experience that oxymyoglobin exposed to mildly acidic conditions becomes ferric and, in the process, undergoes some minor but apparently irreversible change. The chapter also explains the isolation and purification of vertebrate myoglobins.

Facilitated Oxygen Diffusion: THE ROLE OF LEGHEMOGLOBIN IN NITROGEN FIXATION BY BACTEROIDS ISOLATED FROM SOYBEAN ROOT NODULES

Abstract Oxyleghemoglobin, added to well stirred suspensions of bacteroids isolated from soybean root nodules, enhances the rates of oxygen consumption and of reduction of acetylene to ethylene, a measure of the activity of the enzyme complex, nitrogenase. The rate of incorporation of labeled nitrogen from 15N2 into NH3, another measure of nitrogenase activity, is also augmented. Ferric leghemoglobin, as well as ferric horseradish peroxidase and ferric cytochrome c peroxidase, were ineffective. Superoxide dismutase and catalase did not significantly affect the rates of oxygen uptake or acetylene reduction. Each of 12 oxygen-binding proteins (including two non-heme proteins) tested augmented both oxygen up-take and acetylene reduction by bacteroid suspensions; the magnitude of the increments was different for different proteins. We deduce that oxyleghemoglobin and other oxygenbinding proteins exert their effects by facilitating the diffusion of oxygen through a thin layer of solution, the unstirred layer, surrounding the bacteroids. In the absence of oxyleghemoglobin, suspensions of bacteroids maintain a substantial oxygen uptake, which is largely ineffective in supporting nitrogenase activity. The addition of oxyleghemoglobin brings about an increment of oxygen consumption (the oxygen uptake) and promotes nitrogenase activity, which increases roughly 10-fold. The ratio of the increment in oxygen consumption to the increment of acetylene reduction is about 2, corresponding to a calculated P:O ratio for bacteroid oxidative phosphorylation of 1:3. A convenient explanation of our results is that in the absence of leghemoglobin, the ineffective oxygen uptake maintains a very low oxygen pressure at the bacteroid surface. In the presence of leghemoglobin we suggest that the operation of leghemoglobin-facilitated oxygen diffusion increases the po2 at the bacteroid surface. This results in the effective oxygen uptake which supports nitrogenase activity. The possible mode of operation of this control system is discussed.

The Molecular Mechanism of Hemoglobin-facilitated Oxygen Diffusion

Hemoglobin in solution augments the rate of steady state diffusion of oxygen. The flux of oxygen exceeds the diffusive flux by an amount, the facilitated flux, which is constant for a given hemoglobin concentration and is invariant with changing oxygen tension. The facilitated flux is inversely proportional to the length of the diffusion path. Facilitation decreases with increasing molecular size of the oxygen-binding proteins. From this it is concluded that facilitation results from random displacements of oxyhemoglobin molecules. Translational diffusion is considered to make a far larger contribution than rotatory diffusion. Facilitation is proportional to hemoglobin concentration in dilute solutions which approach ideal behavior and declines at greater protein concentration. It persists in very concentrated hemoglobin solutions (34%) in which the hemoglobin molecules are close packed. Facilitation requires no special property of hemoglobin other than reversible oxygen binding since hemerythrin which is not a heme protein facilitates oxygen diffusion. The reactions occurring during facilitated diffusion are shown to be combination and dissociation of hemoglobin and oxygen. Oxygen displacement or transfer reactions are excluded. The rates of chemical reactions may become rate-limiting. The rate-limiting step is a reaction of oxyhemoglobin. Comparison of the rates of facilitation by several hemoglobins suggests that the rate-limiting step is ligand dissociation. Facilitated carbon monoxide diffusion is minute compared to that of oxygen.