Many of the synthetic strategies to create hemoglobin based acellular blood substitutes, employ modifications that perturb the central cavity and extend the hydrodynamic volume. Cross-bridging (cross-linking), effector binding and PEGolation (attachment of polyethylene glycols) result in a wide range of Hbs having a broad spectrum of functional properties. Some of these properties such as enhanced tetramer stability and reduced oxygen affinity are needed attributes for potential blood substitutes. It is our contention that the rational design of this and the next generation of blood substitutes requires an understanding of how these modifications couple to functionally relevant domains of the protein. Four functionally important intra protein communication pathways are proposed. Experiments are being pursued to determine how central cavity modifications and PEGolation affect both key elements of these pathways and the communication between these elements. Several optical spectroscopic tools are to be used to probe the static and dynamic properties of key domains. Visible resonance Raman and near IR absorption will probe the heme and its immediate environment. Steady state front face fluorescence, time correlated single photon counting fluorescence lifetime measurements and nanosecond pulse-probe fluorescence will be used to probe tryptophans, fluorescent analogues of DPG and fluorescent probe labeled beta93. UV resonance Raman will be used both to discriminate between tryptophans and to probe interactions between the A and E helices the alpha1beta2 interface and the interactions between the FG corner and the tyrosines of the C terminus. Iron-metal and meso-proto heme hybrid forms of native, mutant, PEGolated, cross-bridged and pseudo cross-bridged Hb's will be used to create a range of tertiary structures and provide a means of probing subunit specific domains. Subunits with tryptophan replaced with 5-OH tryptophan will be used to provide subunit specificity for the fluorescence studies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058247-02
Application #
2546195
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
1996-09-30
Project End
2001-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Physiology
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Sahu, Ranjit K; Nacharaju, Parimala; Manjula, Belur N et al. (2009) Induced plasma expander-like properties as a function of PEG-chains on extension arm facilitated PEGylation of albumin: ""mushroom to brush-like"" conformational transition of the PEG-albumin conjugate. Artif Cells Blood Substit Immobil Biotechnol 37:245-56
Hu, Tao; Li, Dongxia; Manjula, Belur N et al. (2009) PEGylation of Val-1(alpha) destabilizes the tetrameric structure of hemoglobin. Biochemistry 48:608-16
Li, Dongxia; Hu, Tao; Manjula, Belur N et al. (2009) Extension arm facilitated pegylation of alphaalpha-hemoglobin with modifications targeted exclusively to amino groups: functional and structural advantages of free Cys-93(beta) in the PEG-Hb adduct. Bioconjug Chem 20:2062-70
Meng, Fantao; Manjula, Belur N; Tsai, Amy G et al. (2009) Hexa-thiocarbamoyl phenyl PEG5K Hb: vasoactivity and structure: influence of rigidity of the conjugation linkage on the PEGylation induced plasma expander-like solution properties of PEG-Hb adducts. Protein J 28:199-212
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Meng, Fantao; Manjula, Belur N; Smith, Paul K et al. (2008) PEGylation of human serum albumin: reaction of PEG-phenyl-isothiocyanate with protein. Bioconjug Chem 19:1352-60
Ananda, K; Nacharaju, Parimala; Smith, Paul K et al. (2008) Analysis of functionalization of methoxy-PEG as maleimide-PEG. Anal Biochem 374:231-42
Li, Dongxia; Manjula, Belur N; Ho, Nancy T et al. (2007) Molecular aspects of the high oxygen affinity of non-hypertensive hexa pegylated hemoglobin, [(SP-PEG5K)(6)-Hb]. Artif Cells Blood Substit Immobil Biotechnol 35:19-29
Nacharaju, Parimala; Manjula, Belur N; Acharya, Seetharama A (2007) Thiolation mediated pegylation platform to generate functional universal red blood cells. Artif Cells Blood Substit Immobil Biotechnol 35:107-18
Hu, Tao; Manjula, Belur N; Li, Dongxia et al. (2007) Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin. Biochem J 402:143-51

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