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1.  Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix 
The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin has been solved.
The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128.
doi:10.1107/S1744309113005708
PMCID: PMC3614163  PMID: 23545644
hemoglobin; deer mouse; oxygen affinity; Peromyscus maniculatus
2.  A Long-Acting, MonoPEGylated Human Growth Hormone Analog is a Potent Stimulator of Weight Gain and Bone Growth in Hypophysectomized Rats. 
Endocrinology  2007;148(4):1590-1597.
Recombinant human Growth Hormone (GH) is used to treat growth hormone deficiency in children and adults, and wasting in AIDS patients. GH has a circulating half-life of only a few hours in humans and must be administered to patients by daily injection for maximum effectiveness. Previous studies showed that longer-acting forms of GH could be created by modification of GH with multiple 5 kDa amine-reactive polyethylene glycols (PEGs). Eight of nine lysine residues and the N-terminal amino acid were modified to varying extents by amine-PEGylation of GH. The amine-PEGylated GH product comprised a complex mixture of multiple PEGylated species that differed from one another in mass, in vitro bioactivity and in vivopotency. In vitro bioactivity of GH was reduced 100- to 1,000-fold by extensive amine-PEGylation of the protein. Here we describe a homogeneously modified, monoPEGylated GH protein that possesses near complete in vitro bioactivity, a long half-life and increased potency in vivo. The monoPEGylated GH was created by substituting cysteine for threonine-3 (T3C) of GH, followed by modification of the added cysteine residue with a single 20 kDa cysteine-reactive PEG. The PEG-T3C protein has an approximate 8-fold longer half-life than GH following sc administration to rats. Every other day or every third day administration of PEG-T3C stimulates increases in body weight and tibial epiphysis growth comparable to that produced by daily administration of GH in hypophysectomized rats. Long-acting, monoPEGylated GH analogs such as PEG-T3C are promising candidate for future testing in humans.
doi:10.1210/en.2006-1170
PMCID: PMC1892190  PMID: 17234711
growth hormone; polyethylene glycol; rat; bone; growth
3.  The interaction of 2,3-diphosphoglycerate with various human hemoglobins 
Journal of Clinical Investigation  1970;49(6):1088-1095.
Oxygen equilibria were measured on a number of human hemoglobins, which had been “stripped” of organic phosphates and isolated by column chromatography. In the presence of 2 × 10-4 M 2,3-diphosphoglycerate (2,3-DPG), the P50 of hemoglobins A, A2, S, and C increased about twofold, signifying a substantial and equal decrease in oxygen affinity. Furthermore, hemoglobins Chesapeake and MMilwaukee-1 which have intrinsically high and low oxygen affinities, respectively, also showed a twofold increase in P50 in the presence of 2 × 10-4 M 2,3-DPG. In comparison to these, hemoglobins AIC and F were less reactive with 2,3-DPG while hemoglobin FI showed virtually no reactivity. The N-terminal amino of each β-chain of hemoglobin AIC is linked to a hexose. In hemoglobin FI the N-terminal amino of each γ-chain is acetylated. These results suggest that the N-terminal amino groups of the non-α-chains are involved in the binding of 2,3-DPG to hemoglobin.
PMCID: PMC322576  PMID: 5422014
4.  Oxygen equilibrium of hemoglobin E 
Journal of Clinical Investigation  1972;51(11):2984-2987.
Oxygen equilibrium was determined on hemoglobin of individuals both heterozygous and homozygous for hemoglobin E. The whole blood oxyhemoglobin dissociation curve of AE blood was identical to that of normal AA blood. E hemoglobin, isolated by diethylaminoethyl Sephadex and carboxymethyl cellulose column chromatography, had oxygen affinity, heme-heme interaction, and Bohr effect identical to those of hemoglobin A prepared from the same column. Furthermore, the two hemoglobins had equal reactivity with 2,3-diphosphoglycerate. Phosphate-free hemolysates of blood from E and A homozygotes also had identical oxygen saturation curves. These results do not confirm earlier reports that hemoglobin E has an abnormally low oxygen affinity.
PMCID: PMC292451  PMID: 5080424
5.  Hemoglobin Brigham (α2Aβ2100 Pro→Leu). HEMOGLOBIN VARIANT ASSOCIATED WITH FAMILIAL ERYTHROCYTOSIS 
Journal of Clinical Investigation  1973;52(8):2060-2067.
Erythrocytosis associated with the presence of a hemoglobin with increased oxygen affinity has been reported for 10 hemoglobin variants, most of which demonstrate altered electrophoretic mobility. Several members of a family were found to have erythrocytosis, and both the whole blood and the hemoglobin exhibited increased oxygen affinity. Phosphate-free hemoglobin solutions had a normal Bohr effect and reactivity to 2,3-diphosphoglycerate. The electrophoretic properties of the hemoglobin were normal, but on peptide mapping of a tryptic digest of the isolated β-chains, a normal βT11 peptide and an abnormal βT11 with greater Rf were seen. Analysis of the abnormal peptide showed the substitution of leucine for the normal proline at β100 (helical residue G2).
The hemoglobin variant, designated Hb Brigham, serves to emphasize the necessity for detailed evaluation of the structure and function of hemoglobin in familial erythrocytosis even with electrophoretically “normal” hemoglobin.
PMCID: PMC302488  PMID: 4719677
6.  Heme Degradation and Vascular Injury 
Antioxidants & Redox Signaling  2010;12(2):233-248.
Abstract
Heme is an essential molecule in aerobic organisms. Heme consists of protoporphyrin IX and a ferrous (Fe2+) iron atom, which has high affinity for oxygen (O2). Hemoglobin, the major oxygen-carrying protein in blood, is the most abundant heme-protein in animals and humans. Hemoglobin consists of four globin subunits (α2β2), with each subunit carrying a heme group. Ferrous (Fe2+) hemoglobin is easily oxidized in circulation to ferric (Fe3+) hemoglobin, which readily releases free hemin. Hemin is hydrophobic and intercalates into cell membranes. Hydrogen peroxide can split the heme ring and release “free” redox-active iron, which catalytically amplifies the production of reactive oxygen species. These oxidants can oxidize lipids, proteins, and DNA; activate cell-signaling pathways and oxidant-sensitive, proinflammatory transcription factors; alter protein expression; perturb membrane channels; and induce apoptosis and cell death. Heme-derived oxidants induce recruitment of leukocytes, platelets, and red blood cells to the vessel wall; oxidize low-density lipoproteins; and consume nitric oxide. Heme metabolism, extracellular and intracellular defenses against heme, and cellular cytoprotective adaptations are emphasized. Sickle cell disease, an archetypal example of hemolysis, heme-induced oxidative stress, and cytoprotective adaptation, is reviewed. Antioxid. Redox Signal. 12, 233–248.
doi:10.1089/ars.2009.2822
PMCID: PMC2821146  PMID: 19697995
7.  Single amino acid substitution in important hemoglobinopathies does not disturb molecular function and biological process 
Hemoglobin is an important protein found in the red cells of many animals. In humans, the hemoglobin is mainly distributed in the red blood cell. Single amino acid substitution is the main pathogenesis of most hemoglobin disorders. Here, the author used a new gene ontology technology to predict the molecular function and biological process of four important hemoglobin disorders with single substitution. The four studied important abnormal hemoglobins (Hb) with single substitution included Hb S, Hb E, Hb C, and Hb J-Baltimore. Using the GoFigure server, the molecular function and biological process in normal and abnormal hemoglobins was predicted. Compared with normal hemoglobin, all studied abnormal hemoglobins had the same function and biological process. This indicated that the overall function of oxygen transportation is not disturbed in the studied hemoglobin disorders. Clinical findings of oxygen depletion in abnormal hemoglobin should therefore be due to the other processes rather than genomics, proteomics, and expression levels.
PMCID: PMC2527669  PMID: 18686781
hemoglobin; amino acid; substitution; function
8.  The acetylation of hemoglobin by aspirin. In vitro and in vivo. 
Journal of Clinical Investigation  1975;56(1):201-207.
The chemical modification of hemoglobin by aspirin (ASA) has been studied, both in intact human red cells and in purified hemoglobin solutions. After incubation of red cells with 20 mM [acetyl-1minus14C]ASA, incorporation of radioactivity into hemoglobin was observed in agreement with the results of Klotz and Tam (1973. Proc. Natl. Acad. Sci. U. S. A. 70: 1313-1315). In contrast, no labeling of hemoglobin was seen when [carbosyl-14-C]ASA was used. These results indicate that ASA acetylates hemoglobin. The acetylated hemoglobin was readily separated from unmodified hemoglobin by both gel electrofocusing and by column chromatography. Quantitation of the extent of acetylation by densitometric scanning of gels agreed very well with estimates obtained from radioactivity measurements. Hemolysates prepared from red cells incubated with ASA showed normal oxygen affinity and heme-heme interaction. Purified acetylated hemoglobin had a slightly increased oxygen affinity and decreased heme-heme interaction. There was no difference in the rate of acetylation of oxy- and deoxyhemoglobin. ASA acetylated column-purified hemoglobin A more readily than hemoglobin in crude hemolysate, but less rapidly than purified human serum albumin. The rate of acetylation of hemoglobulin increased with pH up to approximately pH 8,5. Structural studies were done on hemoglobin incubated with 2.0 mM and 20 mM [acetyl-1-14-C]ASA. Alpha- and beta-chains were acetylated almost equally. Tryptic digests of purified acetylated subunits were fingerprinted on cellulose thin layer plates and autoradiographed. Both alpha- and beta-chains showed a number of radioactive spots that were either ninhydrin negative or weakly ninhydrin positive. These results indicate that hemoglobin is acetylated at a number of sites, probably at the epislon-amino group of lysine residues. To determine whether ASA acetylates hemoglobin in vivo, hemolysates of 14 patients on long-term high-dose ASA therapy were analyzed by gel electrofocusing and compared to specimens of individuals not receiving ASA. The ASA-treated group had a twofold increase in a minor hemoglobin component having an isoelectric point lower than that of hemoglobin A, and indistinguishable from the minoe component which appears when hemoglobin is incubated with ASA in vitro.
Images
PMCID: PMC436570  PMID: 237937
9.  The impact of PEGylation patterns on the in vivo biodistribution of mixed shell micelles 
Polyethylene glycol (PEG)-ylation is a widely used strategy to fabricate nanocarriers with a long blood circulation time. Further elaboration of the contribution of the surface PEGylation pattern to biodistribution is highly desirable. We fabricated a series of polyion complex (PIC) micelles PEGylated with different ratios (PEG2k and PEG550). The plasma protein adsorption, murine macrophage uptake, and in vivo biodistribution with iodine-125 as the tracer were systematically studied to elucidate the impact of PEGylation patterns on the biodistribution of micelles. We demonstrated that the PEGylated micelles with short hydrophilic PEG chains mixed on the surface were cleared quickly by the reticuloendothelial system (RES), and the single PEG2k PEGylated micelles could efficiently prolong the blood circulation time and increase their deposition in tumor sites. The present study extends the understanding of the PEGylation strategy to further advance the development of ideal nanocarriers for drug delivery and imaging applications.
doi:10.2147/IJN.S51566
PMCID: PMC3825670  PMID: 24235825
drug delivery; PEGylation; mixed shell micelles; macrophage uptake; in vivo biodistribution
10.  Net charge and oxygen affinity of human hemoglobin are independent of hemoglobin concentration 
The Journal of General Physiology  1978;72(6):765-773.
The dependence of net charge and oxygen affinity of human hemoglobin upon hemoglobin concentration was reinvestigated. In contrast to earlier reports from various laboratories, both functional properties of hemoglobin were found to be independent of hemoglobin concentration. Two findings indicate a concentration-independent net charge of carbonmonoxy hemoglobin at pH 6.6: (A) The pH value of a given carbonmonoty hemoglobin solution remains constant at 6.6 when the hemoglobin concentration is raised from 10 to 40 g/dl, indicating that there is no change in protonation of titratable groups of hemoglobin: (b) the net charge of carbonmonoxy hemoglobin as estimated from the Donnan distribution of 22Na+ shows no dependence on hemoglobin concentration in this concentration range. The oxygen affinity of human hemoglobin was determined from measurements of oxygen concentrations in equilibrated samples using a Lex-O2-Con apparatus (Lexington Instruments, Waltham, Mass.). P50 averaged 11.4 mm Hg at 37 degrees C, pH = 7.2, and ionic strength approximately 0.15. Neither P50 nor Hill's n showed any variation with hemoglobin concentrations increasing from 10 to 40 g/dl.
PMCID: PMC2228487  PMID: 32221
11.  Oxygen equilibrium of hemoglobin J Cape Town 
Journal of Clinical Investigation  1971;50(7):1554-1555.
Polycythemia in carriers of hemoglobin J Cape Town or hemoglobin Chesapeake is thought to be produced by increased oxygen affinity of their blood. Both hemoglobins involve substitution of amino acid residue α FG-4. Measurements reported here, of the oxygen equilibrium of purified hemoglobin J Cape Town, permit direct comparison of the two hemoglobins. J Cape Town exhibits lower oxygen affinity, and greater heme-heme interaction, than Chesapeake; both exhibit normal Bohr effects. Substitution of one polar amino acid residue for another of opposite charge (arginine → glutamic acid) thus appears to create less disruption of the interface between α- and β-chains than substitution of a nonpolar residue (arginine → leucine).
PMCID: PMC292097  PMID: 5090068
12.  Subunit dissociation of certain abnormal human hemoglobins 
Journal of Clinical Investigation  1969;48(1):126-138.
The extent of dissociation of various hemoglobins into subunits was estimated from their elution volumes (Ve) on G-100 Sephadex. Under the same controlled conditions carboxyhemoglobins A, A3 (A1), F, S, and C all had the same elution volumes. The carboxy and cyanmet derivatives of hemoglobin Kansas (a variant with very low oxygen affinity) had a relatively high Ve, indicating a decreased mean molecular weight and therefore an increased tendency to form dimers and even monomers. Conversely, the liganded derivatives of hemoglobin Chesapeake (a variant with high oxygen affinity) had a relatively low Ve, suggestive of an impaired degree of subunit dissociation. Deoxyhemoglobin Chesapeake had a Ve identical with that of deoxyhemoglobin A. Cat hemoglobin, known to have an unusually low oxygen affinity, was found to have a higher Ve than human, dog, rabbit, rat, or guinea pig hemoglobins.
Haptoglobin is thought to bind αβ dimers in preference to the α2β2-tetramer. The comparative haptoglobin affinities of the human hemoglobins were measured by competition between the test hemoglobin and radioactive reference hemoglobin for haptoglobin binding sites. Hemoglobins A, F, S, and C all seemed to bind equally readily, but hemoglobin Kansas and cat hemoglobin showed a higher affinity, and hemoglobin Chesapeake a lower affinity.
These results are in accord with recently proposed models which predict that hemoglobins which have an increased degree of subunit dissociation will have a low oxygen affinity, and vice versa.
PMCID: PMC322198  PMID: 5765016
13.  Hemoglobin substitutes 
European Spine Journal  2004;13(Suppl 1):S76-S82.
Orthopaedic patients frequently require blood transfusions to treat peri-operative anemia. Research in the area of hemoglobin substitutes has been of great interest since it holds the promise of reducing the reliance on allogeneic blood transfusions. The three categories of hemoglobin substitutes are (1) cell-free, extracellular hemoglobin preparations made from human or bovine hemoglobin (hemoglobin-based oxygen carriers or HBOCs); (2) fluorine-substituted linear or cyclic carbon chains with a high oxygen-carrying capacity (perfluorocarbons); and (3) liposome-encapsulated hemoglobin. Of the three, HBOCs have been the most extensively studied and tested in preclinical and clinical trials that have shown success in diminishing the number of blood transfusions as well as an overall favorable side-effect profile. This has been demonstrated in vascular, cardiothoracic, and orthopaedic patients. HBOC-201, which is a preparation of cell-free bovine hemoglobin, has been approved for clinical use in South Africa. These products may well become an important tool for physicians treating peri-operative anemia in orthopaedic patients.
doi:10.1007/s00586-004-0737-x
PMCID: PMC3592184  PMID: 15168238
Hemoglobin substitute; HBOC; HBOC-201; Perfluorocarbons
14.  PEGylation of interleukin-10 for the mitigation of enhanced pain states 
The anti-inflammatory cytokine interleukin-10 (IL-10) shows promise for the treatment of neuropathic pain, but for IL-10 to be clinically useful as a short-term therapeutic its duration needs to be improved. In this study, IL-10 was covalently modified with polyethylene glycol (PEG) with the goal of stabilizing and increasing protein levels in the CSF to improve the efficacy of IL-10 for treating neuropathic pain. Two different PEGylation methods were explored in vitro to identify suitable PEGylated IL-10 products for subsequent in vivo testing. PEGylation of IL-10 by acylation yielded a highly PEGylated product with a 35-fold in vitro biological activity reduction. PEGylation of IL-10 by reductive amination yielded products with a minimal number of PEG molecules attached and in vitro biological activity reductions of ~3-fold. In vivo collections of cerebrospinal fluid after intrathecal administration demonstrated that 20 kDa PEG attachment to IL-10 increased the concentration of IL-10 in the cerebrospinal fluid over time. Relative to unmodified IL-10, the 20 kDa PEG-IL-10 product exhibited an increased therapeutic duration and magnitude in an animal model of neuropathic pain. This suggests that PEGylation is a viable strategy for the short-term treatment or, in conjunction with other approaches, the long-term treatment of enhanced pain states.
doi:10.1002/jbm.a.32611
PMCID: PMC3604983  PMID: 19768789
glia; interleukin-10; intrathecal delivery; neuropathic pain; PEGylation
15.  The Relationships between Arterial Oxygen Flow Rate, Oxygen Binding by Hemoglobin, and Oxygen Utilization after Myocardial Infarction 
Journal of Clinical Investigation  1974;54(3):501-513.
The interrelationships of arterial oxygen flow rate index, oxygen binding by hemoglobin, and oxygen consumption have been examined in patients with acute myocardial infarction. Proportional extraction of oxygen increased in close association with decreasing oxygen flow rate, and hence, whole body oxygen consumption was constant over nearly a three-fold variation in arterial oxygen flow rate. A reduction in hemoglobin-oxygen affinity at in vivo conditions of pH. Pco2 and temperature also occurred in proportion to the reduction in arterial oxygen flow rate. Therefore, the increased proportional removal of oxygen from arterial blood at low oxygen flow rates, required to maintain oxygen consumption, may have been facilitated by the reduced affinity of hemoglobin for oxygen at in vivo conditions. However, the decrease in affinity did not appear to explain more than 30-40% of the increased extraction.
Respiratory alkalosis was a frequent occurrence in these patients and 2,3-diphosphoglycerate was positively associated with blood pH as well as with the time-averaged proportion of deoxyhemoglobin in arterial and venous blood.
Hemoglobin-oxygen affinity measured at standard conditions and the mixed venous oxygen saturation were equally good indicators of reduced arterial oxygen flow rate in patients without shock. However, S̄vo2 is more easily measured and is a more useful indicator of reduced oxygen flow rate, since its relationship to oxygen flow appears to be independent of affinity changes and time.
PMCID: PMC301582  PMID: 4855047
16.  Metabolically Stabilized Long-Circulating PEGylated Polyacridine Peptide Polyplexes Mediate Hydrodynamically Stimulated Gene Expression in Liver 
Gene therapy  2010;18(1):23-37.
A novel class of PEGylated polyacridine peptides was developed that mediate potent stimulated gene transfer in the liver of mice. Polyacridine peptides, (Acr-X)n-Cys-PEG, possessing 2–6 repeats of Lys-acridine (Acr) spaced by either Lys, Arg, Leu or Glu, were Cys derivatized with polyethylene glycol (PEG 5000 Da) and evaluated as in vivo gene transfer agents. An optimal peptide of (Acr-Lys)6-Cys-PEG was able to bind to plasmid DNA (pGL3) with high affinity by polyintercalation, stabilize DNA from metabolism by DNAse and extend the pharmacokinetic half-life of DNA in the circulation for up to 2 hrs. A tail vein dose of PEGylated polyacridine peptide pGL3 polyplexes (1 μg in 50 μl), followed by a stimulatory hydrodynamic dose of normal saline at times ranging from 5–60 min post-DNA administration, led to a high level of luciferase expression in the liver, equivalent to levels mediated by direct hydrodynamic dosing of 1 μg of pGL3. The results establish the unique properties of PEGylated polyacridine peptides as a new and promising class of gene delivery peptides that facilitate reversible binding to plasmid DNA, protecting it from DNase in vivo resulting in an extended circulatory half-life, and release of transfection-competent DNA into the liver to mediate a high-level of gene expression upon hydrodynamic boost.
doi:10.1038/gt.2010.117
PMCID: PMC2990782  PMID: 20720577
Gene Delivery; DNA pharmacokinetics; peptide; gene expression; biodistribution
17.  PLASMA SUBSTITUTES  
Hemoglobin and globin alone, supplemented, or modified in various ways are seriously considered as plasma substitutes. Human globin given to doubly depleted (anemic and hypoproteinemic) dogs by vein contributes to the production of new hemoglobin and plasma protein, but there is some toxicity and weight loss. Dog hemoglobin given intraperitoneally is better tolerated and somewhat more completely utilized with more blood proteins formed and less weight loss. Dog globin (tryptic digest) given by vein in anemic dogs is associated with a moderate production of new hemoglobin. Horse globin by mouth contributes to the formation of new hemoglobin in the standard anemic dog. Dog hemoglobin given intraperitoneally in protein fasting, non-anemic dogs is well utilized to maintain nitrogen and weight balance. A dl-isoleucine supplement fails to improve this utilization of hemoglobin for maintenance in the dog. A small supplement of dl-methionine greatly improves the utilization of dog hemoglobin for maintenance in the dog and further addition of isoleucine is without effect. The intermediary metabolism of dog hemoglobin is not yet worked out. Electrophoretic analyses (Table 6) suggest that globin appears in the peripheral circulation after intraperitoneal injections of hemoglobin.
PMCID: PMC2135595  PMID: 19871535
18.  Hemoglobin Yakima: II. High Blood Oxygen Affinity Associated with Compensatory Erythrocytosis and Normal Hemodynamics* 
Journal of Clinical Investigation  1967;46(11):1848-1854.
Erythrocytosis without clinical illness was noted in a man and his two daughters. Their blood contained approximately 62% hemoglobin A and 38% a new hemoglobin, designated hemoglobin Yakima. The oxygen affinity of whole blood from each subject was greatly increased and heme-heme interactions were impaired. At 37°C and a plasma pH of 7.40, the oxygen pressure required to produce 50% saturation of hemoglobin with oxygen was only 12 mm Hg as compared with a normal of 26 mm Hg. The high oxygen affinity of this blood is attributed to the presence of hemoglobin Yakima; and the increased oxygen affinity was shown to be characteristic of the isolated abnormal hemoglobin. A Bohr effect was present in hemoglobin Yakima.
Arterial oxygen pressure, oxygen consumption, and cardiac output at rest were normal. With respect to oxygen delivery to tissues, the increased hemoglobin concentration appears to be the major compensation for the marked displacement of the oxygen-hemoglobin equilibrium curve, although other factors may contribute. The finding of high normal quantities of erythropoietin in the urine is consistent with this degree of erythrocytosis.
PMCID: PMC292935  PMID: 6061752
19.  N-PEGylation of a Reverse Turn is Stabilizing in Multiple Sequence Contexts unlike N-GlcNAcylation 
ACS chemical biology  2011;6(11):1188-1192.
The intrinsic stabilization of therapeutic proteins by N-glycosylation can endow them with increased shelf and serum half-lives owing to lower populations of misfolded and unfolded states, which are susceptible to aggregation and proteolysis. Conjugation of polyethylene glycol (PEG) oligomers to nucleophilic groups on the surfaces of folded proteins (i.e., PEGylation) is a chemical alternative to N-glycosylation, in that it can also enhance the pharmacologic attributes of therapeutic proteins. However, the energetic consequences PEGylation are currently not predictable. We find that PEGylation of an Asn residue in reverse turn 1 of the Pin WW domain is intrinsically stabilizing in several sequence contexts, unlike N-glycosylation, which is only stabilizing in a particular sequence context. Our thermodynamic data are consistent with the hypothesis that PEGylation destabilizes the protein denatured state ensemble via an excluded volume effect, whereas N-glycosylation-associated stabilization results primarily from native state interactions between the N-glycan and the protein.
doi:10.1021/cb200277u
PMCID: PMC3220771  PMID: 21939258
20.  Insensitivity of cerebral oxygen transport to oxygen affinity of hemoglobin-based oxygen carriers 
Biochimica et biophysica acta  2008;1784(10):1387-1394.
The cerebrovascular effects of exchange transfusion of various cell-free hemoglobins that possess different oxygen affinities are reviewed. Reducing hematocrit by transfusion of a non-oxygen-carrying solution dilates pial arterioles on the brain surface and increases cerebral blood flow to maintain a constant bulk oxygen transport to the brain. In contrast, transfusion of hemoglobins with P50 of 4–34 Torr causes constriction of pial arterioles that offsets the decrease in blood viscosity to maintain cerebral blood flow and oxygen transport. The autoregulatory constriction is dependent on synthesis of 20-HETE from arachidonic acid. This oxygen-dependent reaction is apparently enhanced by facilitated oxygen diffusion from the red cell to the endothelium arising from increased plasma oxygen solubility in the presence of low or high-affinity hemoglobin. Exchange transfusion of recombinant hemoglobin polymers with P50 of 3 and 18 Torr reduces infarct volume from experimental stroke. Cell-free hemoglobins do not require a P50 as high as red blood cell hemoglobin to facilitate oxygen delivery.
doi:10.1016/j.bbapap.2008.01.001
PMCID: PMC2562895  PMID: 18230370
Anemia; Blood substitute; Cerebral blood flow; Hemoglobin; Oxygen affinity; Oxygen transport
21.  Targeted O2 delivery by blood substitutes:in vitroarteriolar simulations of first- and second-generation products 
Microvascular research  2008;76(3):169-179.
The O2 transport from mixtures of commercially produced hemoglobin-based O2 carriers (HBOCs) and red blood cells (RBCs) flowing through arteriolar-sized (25-μm) conduits is simulated. A generalized treatment of extraluminal O2 transport processes is used to reflect variations in physiological conditions, such as increased O2 consumption. Of the HBOCs considered, polymerized bovine hemoglobin (PolyBvHb, p50 = 54 mmHg), tetrameric cross-linked human hemoglobin (ααHb, p50 = 33 mmHg), and PEGylated human hemoglobin (MP4, p50 = 5 mmHg), only MP4 does not increase O2 extraction ratios when compared to RBC suspensions alone. A reduction in arteriolar O2 extraction is likely to be beneficial for HBOCs by preventing O2-induced vasoactivity and maximizing the supply of O2 available to the capillaries. Results from in vivo HBOC transfusion experiments cannot be predicted by the model, unless PolyBvHb has a significant decrease in extraluminal O2 transport resistance as compared to MP4. This result is consistent with the literature that shows arteriolar O2 consumption to increase with intravascular pO2.
doi:10.1016/j.mvr.2008.07.002
PMCID: PMC2593130  PMID: 18671987
Facilitated diffusion; O2 affinity; transport simulation; vasoconstriction; blood substitute
22.  Blood substitutes: Haemoglobin therapeutics in clinical practice 
Critical Care  1999;3(5):R99-R102.
Early approaches to the development of oxygen carriers involved the use of stroma-free hemoglobin solutions. These solutions did not require blood typing or crossmatching and could be stored for long periods. In addition, a variety of methods have been developed in chemically modifying and stabilizing the hemoglobin molecule. Several hemoglobin therapeutics are now in clinical trials as temporary alternatives to blood or as therapeutic agents for ischemia. The various hemoglobin products under development are derived from three principal sources: human, bovine and genetically engineered hemoglobin. Diaspirin cross-linked hemoglobin (DCLHb), administered at doses ranging from approximately 20-1000 ml, has been investigated in a number of clinical trials in patients undergoing orthopedic, abdominal aortic repair, major abdominal surgery, cardiac surgery and in critically ill patients with septic shock. In several studies, DCLHb was effective in avoiding the transfusion. However, Baxter Healthcare Corporation (Chicago, Illinois, USA) stopped the development of DCLHb after two unsuccessful trials in trauma patients. Bovine polymerized hemoglobin has also been extensively studied. Several phase II and phase III trials have been performed with this product in hemorrhagic surgery, cardiac surgery and vascular surgery, but data have not yet been published. Hemoglobin therapeutics could provide an important new option as an alternative to blood transfusion. Furthermore, they may be able to provide an immediate on-site replacement for traumatic blood loss, prevent global ischemia and organ failure, treat focal ischemia, and provide effective hemodynamic support for septic shock-induced hypotension.
doi:10.1186/cc365
PMCID: PMC137240  PMID: 11094489
hemoglobin-based oxygen carriers; trauma; avoidance of transfusion; surgical setting; critically ill patients
23.  Salvage of focal cerebral ischemic damage by transfusion of high O2-affinity recombinant hemoglobin polymers in mouse 
Cell-free hemoglobin solutions with high oxygen affinity might be beneficial for selectively delivering oxygen to ischemic tissue. A recombinant hybrid hemoglobin molecule was designed using the human α-subunit and the bovine β-subunit, with placement of surface cysteines to permit disulfide bond polymerization of the tetramers. The resulting protein generated from an Escherichia coli expression system had a molecular mass >1 MDa, a P50 of ~3 Torr, and a cooperativity of n = 1.0. Anesthetized mice were transfused during 2-h occlusion of the middle cerebral artery. Compared with transfusion with 5% albumin, cerebral infarct volume was reduced by 41% with transfusion of a 3% solution of the high oxygen-affinity hemoglobin polymer and by 50% with transfusion of a 6% solution of the polymer. Transfusion of a 6% solution of a 500-kDa polymer possessing a P50 of 17 Torr and a cooperativity of n = 2.0 resulted in a 66% reduction of infarct volume. These results indicate that cell-free Hb polymers with P50 values much lower than that of red blood cell hemoglobin are highly capable of salvaging ischemic brain. The assumption that the P50 of blood substitutes should be similar to that of blood might not be warranted when used during ischemic conditions.
doi:10.1152/japplphysiol.01374.2005
PMCID: PMC1764455  PMID: 16424069
blood substitute; cerebral ischemia; middle cerebral artery; oxygen carrier
24.  A combined top-down and bottom-up MS approach for the characterization of hemoglobin variants in Rhesus monkeys 
Proteomics  2010;10(20):3657-3668.
Sickle cell disease is caused by one of the 1200 known hemoglobin variations. A single point mutation β6(A3)Glu→Val leads to sickling of red blood cells, which in turn causes a lack of oxygen supply to tissue and organs. Although sickle cell disease is well understood, treatment options are currently underdeveloped. The only FDA approved drug is hydroxyurea, an inducer of fetal γ-hemoglobin, which is known to have a higher oxygen affinity than adult hemoglobins and thus alleviates symptoms. In the search for better cures, Rhesus monkeys (Macaca mulatta) serve as models for monitoring success of induction of fetal γ-hemoglobins and with recent advances in proteomics, mass spectrometry has become the leading technique to determine globin expression. Similar to humans, Rhesus monkeys possess hemoglobin variants that have not been sufficiently characterized to initiate such a study. Therefore, we developed a combined bottom-up and top-down approach to identify and characterize novel hemoglobin variants of the umbilical cord blood of Rhesus monkeys. A total of four different variants were studied: α, β, γ1 and γ2. A new α- and β- hemoglobin variant was identified, and the two previously hypothesized γ-hemoglobins were identified. In addition, glutathionylation of both γ-hemoglobin variants at their cysteines has been characterized. The combined approach outperformed either bottom-up or top-down alone and can be used for characterization of unknown hemoglobin variants and their posttranslational modifications.
doi:10.1002/pmic.201000161
PMCID: PMC3036950  PMID: 20848672
Top down; bottom up; Hemoglobin; variant; glutathionylation; Rhesus monkey
25.  Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC) 
Background
Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable.
Methods
The preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo.
Results
Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 μM from ~170 μM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared.
Conclusion
Valuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.
doi:10.1186/1475-2867-9-9
PMCID: PMC2679726  PMID: 19374748

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