Celia Bonaventura

Fluorescence and oxygen evolution from Chlorella pyrenoidosa

Abstract The process of photosynthetic energy conversion in Chlorella pyrenoidosa was investigated by simultaneous measurement of transient and steady-state rates of O2 evolution and fluorescence. 1. 1. Alternation or superimposition of light 1 and light 2 illumination induces both fast and slow changes in fluorescence and rate of O2 evolution. The fast changes are ascribed to changes in conditions of the reaction centers in the context of the Hill-Bendall 1 model and the kinetic analysis of Eley and Myers 2. The slow changes are interpreted as adaptations to the intensity and wavelength of illumination. The adaptive mechanism is described in terms of slow variation in fraction (α) of total absorbed quanta delivered to System 2. At low intensities, the calculated value of α for cells adapted to light 2 illumination (light 2 state) is approx. 0.9 of α for cells adapted to light 1 illumination (light 1 state). 2. 2. An increase in fluorescence yield was found to accompany the decrease in O2 yield at the onset of light saturation with either light 1 or light 2 excitation. Variation in α is proposed to account for the differences between the maximum fluorescence yield observed in steady-state conditions and the 1.5 times higher maximum yield observed in transient conditions or in cells inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. Variation in α can also explain the observation of a higher rate of fluorescence emission with light 1 excitation than with light 2 excitation for a given steady-state rate of O2 evolution. 3. 3. A model for energy conversion by System 2 is proposed to account for our observations. The model proposes competitive dissipation of absorbed energy by photochemical trapping at reaction centers and by fluorescence and radiationless de-excitation from both the pigment bed and reaction centers of System 2.

S-Nitrosohemoglobin Is Unstable in the Reductive Erythrocyte Environment and Lacks O2/NO-linked Allosteric Function

Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as anin vivo reservoir for NO from which NO release is allosterically linked to oxygen release. We show here that SNO-Hb undergoes reductive decomposition in erythrocytes, whereas it is stable in purified solutions and in erythrocyte lysates treated with an oxidant such as ferricyanide. Using an extensively validated methodology that eliminates background nitrite and stabilizes erythrocyte S-nitrosothiols, we find the levels of SNO-Hb in the basal human circulation, including red cell membrane fractions, were 46 ± 17 nm in human arterial erythrocytes and 69 ± 11 nm in venous erythrocytes, incompatible with the postulated reservoir function of SNO-Hb. Moreover, we performed experiments on human red blood cells in which we elevated the levels of SNO-Hb to 10,000 times the normal in vivo levels. The elevated levels of intra-erythrocytic SNO-Hb fell rapidly, independent of oxygen tension and hemoglobin saturation. Most of the NO released during this process was oxidized to nitrate. A fraction (25%) was exported as S-nitrosothiol, but this fraction was not increased at low oxygen tensions that favor the deoxy (T-state) conformation of Hb. Results of these studies show that, within the redox-active erythrocyte environment, the β-globin cysteine 93 is maintained in a reduced state, necessary for normal oxygen affinity, and incapable of oxygen-linked NO storage and delivery.

Keyhole limpet hemocyanin: Structural and functional characterization of two different subunits and multimers☆

Keyhole limpet hemocyanin (KLH), the large respiratory glycoprotein from the primitive gastropod mollusc, Megathura crenulata, is a potent immunogen used classically as a carrier protein for haptens and more recently in human vaccines and for immunotherapy of bladder cancer. Two KLH isoforms were identified and isolated by high-performance anion exchange chromatography. Subsequent analyses disclosed that these isoforms--designated KLH-A and KLH-B--were composed of distinct subunits that differed in primary structure, molecular weight (KLH-A was 449,000 and KLH-B was 392,000), polymerization/reassociation characteristics, and O2-binding constants (KLH-A had a P50 of 7.32 and KLH-B had a P50 of 2.46). Both subunits appear to be composed of eight oxygen binding domains, and reassociate in solution only with like subunits. These results support the concept that structural and functional heterogeneity is a common feature of molluscan hemocyanins, and provide a rational basis for studying and optimizing the immunostimulatory properties of KLH.

Structural basis for the root effect in haemoglobin.

The remarkable ability of Root effect haemoglobins to pump oxygen against high O2 gradients results from extreme, acid-induced reductions in O2 affinity and cooperativity. The long-sought mechanism for the Root effect, revealed by the 2 Å crystal structure of the ligand-bound haemoglobin from Leiostomus xanthurus at pH 7.5, unexpectedly involves modulation of the R-state. Key residues strategically assemble positive-charge clusters across the allosteric β1β2-interface in the R-state. At low βH, protonation of the βN terminus and His 147(HC3)β within these clusters is postulated to destabilize the R-state and promote the acid-triggered, allosteric R→T switch with concomitant O2 release. Surprisingly, a set of residues specific to Root effect haemoglobins recruit additional residues, conserved among most haemoglobins, to produce the Root effect.

Hemoglobins of two terebellid polychaetes: Enoplobranchus sanguineus and Amphitrite ornata

1. 1. The intracellular, coelomic hemoglobins of the terebellid annelids Enoplobranchus sanguineus and Amphitrite ornata are momoneric proteins possessing high O2 affinities (half-saturation O2 tension, P50 = 1.4 and 2.8 mm, respectively, at 20°C), and lacking homotropic or heterotropic interactions in O2 binding. These O2 affinities are high relative to extracellular vascular hemoglobins, but low relative to mammalian myoglobins, due to considerably higher rates of O2 dissociation. 2. 2. The extracellular, multi-subunit hemoglobin (erythrocruorin) from the vascular system of A. ornata has a low O2 affinity (P50 about 11 mm Hg at 20°C) and shows significant heme-heme interaction (Hills constant n50 = 1.5–2.0). It lacks a Bohr effect between pH 5.8 and 8.0, but shows a large increase in O2 affinity above pH 8.0 that may be due to dissociation into high affinity subunits. 3. 3. The amino acid composition and the amino acid sequence for the first 29 residues of intracellular E. sanguineus hemoglobin show a surprising lack of homology with other simple heme proteins.

Effects of S-nitrosation on oxygen binding by normal and sickle cell hemoglobin.

S-Nitrosated hemoglobin (SNO-Hb) is of interest because of the allosteric control of NO delivery from SNO-Hb made possible by the conformational differences between the R- and T-states of Hb. To better understand SNO-Hb, the oxygen binding properties of S-nitrosated forms of normal and sickle cell Hb were investigated. Spectral assays and electrospray ionization mass spectrometry were used to quantify the degree ofS-nitrosation. Hb A0 and unpolymerized Hb S exhibit similar shifts toward their R-state conformations in response to S-nitrosation, with increased oxygen affinity and decreased cooperativity. Responses to 2,3-diphosphoglycerate were unaltered, indicating regional changes in the deoxy structure of SNO-Hb that accommodate NO adduction. A cycle of deoxygenation/reoxygenation does not cause loss of NO or appreciable heme oxidation. There is, however, appreciable loss of NO and heme oxidation when oxygen-binding experiments are carried out in the presence of glutathione. These results indicate that the in vivo stability of SNO-Hb and its associated vasoactivity depend on the abundance of thiols and other factors that influence transnitrosation reactions. The increased oxygen affinity and R-state character that result fromS-nitrosation of Hb S would be expected to decrease its polymerization and thereby lessen the associated symptoms of sickle cell disease.

HEMOGLOBINS FROM TROUT - STRUCTURAL AND FUNCTIONAL PROPERTIES

SummaryThe diversity of the structural and functional properties of the various components of trout blood may be taken as a type case of molecular adaptation to physiological requirements. Studies on this system yield, in addition, information which appears relevant to the interpretation of the behavior of mammalian hemoglobins.

Urea Tolerance as a Molecular Adaptation of Elasmobranch Hemoglobins

Urea is maintained at moderately high concentrations in the blood and tissues of marine elasmobranchs. Functional properties of the hemoglobins fromn several elasmobranch species are unaffected by urea concentrations as high as 5 molar. This in. sensitivity to urea, which is not observed with human hemoglobin, is accompanied by an increased sensitivity to sodium chloride.

Consequences of chemical modifications on the free radical reactions of human hemoglobin

Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that HBOCs of specific types differ in their ability to generate or interact with free radicals. The differences do not correlate with oxygen affinity. Detailed comparisons with unmodified human hemoglobin, HbA0, were carried out with two cross-linked derivatives: HbA-FMDA, produced by the reaction of human oxyhemoglobin with fumaryl monodibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyhemoglobin with bis(3,5-dibromosalicyl) fumarate. Both derivatives had lower oxygen affinity than unmodified HbA0. As previously reported, exposure of oxyhemoglobin to H2O2 causes generation of free radicals capable of generating formaldehyde from dimethyl sulfoxide. Relative to the reaction catalyzed by 50 microM HbA (18.0 +/- 3.5 nmol/30 min/ml), the formaldehyde formation was roughly 70% for HbA-DBBF and 50% for HbA-FMDA under comparable conditions. More profound differences are exhibited at lower hemoglobin concentrations. Spectral changes of the HBOCs during the reaction differ qualitatively and occur at different rates. The HBOCs also differ in rates of hemoglobin-catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. These results show that stereochemical differences brought about by chemical cross-linking alter the ability of HBOCs to generate radicals and to react with activated oxygen species. These studies also show that the ability of hemoglobin to produce activated species of oxygen can be enhanced or suppressed independently of oxygen affinity.

The role of coelomic and vascular hemoglobin in the annelid family terebellidae

Abstract 1. 1. Monomeric hemoglobins extracted from terebellid coelomic cells have high oxygen affinities (1–3 mm Hg) with no co-operativity or phosphate dependence in oxygen binding. Hill plots describing deoxygenation of hemoglobins within cells have similar P 50 s, but the curves are biphasic. 2. 2. The high molecular weight hemoglobins dissolved in blood have a relatively low oxygen affinity (10 mm Hg) with slight co-operativity and no sensitivity to phosphates and NaCl. In vivo they transport oxygen primarily to coelomic fluid rather than deep tissue. 3. 3. Terebellid hemoglobins are responsible for 30–55 per cent of total oxygen consumption at p O 2 s prevailing both at high tide, when oxygen transport is believed to be their major function, and at low tide when oxygen storage predominates.