Conjoined Hemoglobins. Loss of Cooperativity and Protein−Protein Interactions

Gourianov, Nikolai; Kluger, Ronald

doi:10.1021/bi0513812

Cited by 17 publications

(30 citation statements)

References 23 publications

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“…Reagent 1, with a central sulfone, gave the highest yield of a ∼128 kDa material (Table 1). Globin chain analysis (32) through C4 reverse-phase HPLC showed that only one component derived from native hemoglobin had been modified (Figure 2 (20) and counterparts isophthalyl phosphastes (23,24) that produced cross-linked hemoglobin bis-tetramers.…”

Section: Resultsmentioning

confidence: 99%

Functional Cross-Linked Hemoglobin Bis-tetramers: Geometry and Cooperativity

Kluger

2008

Biochemistry

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Hemoglobin-based oxygen carriers have been sought as stable, sterile alternatives to red cells in transfusions. Problems in clinical trials using cross-linked tetramers have led to proposals that larger assemblies of tetramers may alleviate some of the problems. A study of such assemblies requires materials with defined structures and physical properties. Evaluation of cross-linked bis-tetramers with inflexible linear links between the tetramers revealed that these have very low cooperativity in oxygen binding and would thus be inefficient as oxygen carriers. New, more flexible reagents were designed to cross-link and connect tetramers in two modes: with angular connectors that permit torsional movement (1-3) and with linear connectors that resemble previously studied systems (4-6). The resulting cross-linked bis-tetramers were produced in high yield and were isolated and characterized. Digest mapping showed that modifications were specifically introduced as expected at amino groups in the 2,3-bisphosphoglycerate binding sites within beta subunits. Circular dichroism showed that the secondary structure of the globin chains is maintained while the microenvironment of the hemes is altered. The bis-tetramers derived from 1-3 have oxygen affinity (P(50) = 3.6-4.7) and cooperativity (n(50) = 2.2-2.7) that appear to be suitable for efficient oxygen delivery to hypoxic regions along with increased mass that is expected to minimize extravasation.

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Section: Resultsmentioning

confidence: 99%

Functional Cross-Linked Hemoglobin Bis-tetramers: Geometry and Cooperativity

Kluger

2008

Biochemistry

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“…This has previously been achieved in our lab (71)(72)(73). Tetrafunctional cross-linkers react selectively within hemoglobin tetramers while also creating inter-protein linkages between tetramers (71-73) to give cross-linked bis-tetramers of hemoglobin (BT-Hb) (Scheme 3.1).…”

Section: Discussionmentioning

confidence: 97%

“…In our design for functional bis-tetramers, we have found that the structure of the inter-protein linkage between cross-linked hemoglobin tetramers affects cooperativity (71,73). Linkages with torsional flexibility from a tetrahedral centre, such as is found in a sulfone or ether linkage, give materials with much higher Hill coefficients (n 50 = 2.5- partial pressure between the high P O2 of blood and the low P O2 of tissues.…”

Section: Favourable Oxygen Binding Propertiesmentioning

confidence: 99%

Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives

Lui

Kluger

2009

Biochemistry

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The clinical evaluation of stabilized tetrameric hemoglobin as alternatives to red cells revealed that the materials caused significant increases in blood pressure and related problems and this was attributed to the scavenging of nitric oxide and extravasation. The search for materials with reduced vasoactivity led to the report that conjugates of hemoglobin tetramers and polyethylene glycol (PEG) chains did not elicit these pressor effects. However, this material does not deliver oxygen efficiently due to its lack of cooperativity and high oxygen affinity, making it unsuitable as an oxygen carrier. It has been recently reported that PEGconjugated hemoglobin converts nitrite to nitric oxide at a faster rate than does the native protein, which may compensate for the scavenging of nitric oxide. It is therefore important to alter hemoglobin in order to enhance nitrite reductase activity while retaining its ability to deliver oxygen. If the beneficial effect of PEG is associated with the increased size reducing extravasation, this can also be achieved by coupling cross-linked tetramers to one another, giving materials with appropriate oxygen affinity and cooperativity for use as circulating oxygen carriers. In the present study it is shown that cross-linked bis-tetramers with good oxygen delivery potential have enhanced nitrite reductase activity with k obs = 0.70 M -1 s -1 (24 °C), compared to native protein and cross-linked tetramers, k obs = 0.25 M -1 s -1 and k obs = 0.52 M -1 s -1 , respectively, but are less active in reduction of nitrite than Hb-PEG5K 2 (k obs = 2.5 M -1 s -1 ). However, conjugation of four PEG chains to the bis-tetramer (at each β-Cys-93) produces a material with greatly increased nitrite reductase activity (k obs = 1.8 M -1 s -1 ) while retaining cooperativity (P 50 = 4.1, n 50 = 2.4). Thus, PEGylated bistetramers combine increased size and enhanced nitrite reductase activity expected for decreased vasoactivity with characteristics of an acceptable HBOC.

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“…The binding of a single molecule of dopamine to any of the four unoccupied D2 receptors exerts a negative effect on the other three receptors, lowering their affinity for dopamine. (The situation is analogous to that for hemoglobin where the hemoglobin chains interact to alter the affinities for oxygen; Gourianov and Kluger, 2005.) However, in striatal tissues from animals that are supersensitive to dopamine, the factors contributing to dopamine supersensitivity would reduce the negative interaction between the D2 receptors.…”

Section: The Physical Existence Of the D2 High Statementioning

confidence: 99%

Psychosis pathways converge via D2High dopamine receptors

Seeman

Schwarz

Chen

et al. 2006

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The objective of this review is to identify a target or biomarker of altered neurochemical sensitivity that is common to the many animal models of human psychoses associated with street drugs, brain injury, steroid use, birth injury, and gene alterations. Psychosis in humans can be caused by amphetamine, phencyclidine, steroids, ethanol, and brain lesions such as hippocampal, cortical, and entorhinal lesions. Strikingly, all of these drugs and lesions in rats lead to dopamine supersensitivity and increase the high-affinity states of dopamine D2 receptors, or D2High, by 200-400% in striata. Similar supersensitivity and D2High elevations occur in rats born by Caesarian section and in rats treated with corticosterone or antipsychotics such as reserpine, risperidone, haloperidol, olanzapine, quetiapine, and clozapine, with the latter two inducing elevated D2High states less than that caused by haloperidol or olanzapine. Mice born with gene knockouts of some possible schizophrenia susceptibility genes are dopamine supersensitive, and their striata reveal markedly elevated D2High states; suchgenes include dopamine-beta-hydroxylase, dopamine D4 receptors, G protein receptor kinase 6, tyrosine hydroxylase, catechol-O-methyltransferase, the trace amine-1 receptor, regulator of G protein signaling RGS9, and the RIIbeta form of cAMP-dependent protein kinase (PKA). Striata from mice that are not dopamine supersensitive did not reveal elevated D2High states; these include mice with knockouts of adenosine A2A receptors, glycogen synthase kinase GSK3beta, metabotropic glutamate receptor 5, dopamine D1 or D3 receptors, histamine H1, H2, or H3 receptors, and rats treated with ketanserin or aD1 antagonist. The evidence suggests that there are multiple pathways that convergetoelevate the D2High state in brain regions and that this elevation may elicit psychosis. This proposition is supported by the dopamine supersensitivity that is a common feature of schizophrenia and that also occurs in many types of genetically altered, drug-altered, and lesion-altered animals. Dopamine supersensitivity, in turn, correlates with D2High states. The finding that all antipsychotics, traditional and recent ones, act on D2High dopamine receptors further supports the proposition.

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Conjoined Hemoglobins. Loss of Cooperativity and Protein−Protein Interactions

Cited by 17 publications

References 23 publications

Functional Cross-Linked Hemoglobin Bis-tetramers: Geometry and Cooperativity

Functional Cross-Linked Hemoglobin Bis-tetramers: Geometry and Cooperativity

Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives

Psychosis pathways converge via D2High dopamine receptors

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