US EPA

1097774 

Journal Article 

Chemical characterization of diaspirin cross-linked hemoglobin polymerized with poly(ethylene glycol) 

Buehler, PW; Boykins, RA; Norris, S; Alayash, AI 

2006 

Yes 

Analytical Chemistry
ISSN: 0003-2700
EISSN: 1520-6882 

78 

13 

4634-4641 

English 

16808476 

10.1021/ac060188q 

WOS:000238665200054 

A lack of specificity associated with chemical modification methods used in the preparation of certain hemoglobin (Hb)-based oxygen carriers (HBOCs) may alter Hb structure and function, as amino acids located in critical regions (e.g., alpha-beta interfaces and the 2,3-DPG binding pocket) may unintentionally be targeted. Hb protein surface modifications with various poly(ethylene glycol) (PEG) derivatives have been used as conjugating and polymerizing agents with the intent of improving reaction site specificity/reproducibility and ultimately reducing the untoward hypertensive response due to nitric oxide scavenging by smaller molecular size tetrameric species (i.e., 64 kDa) in HBOC solutions. Previous experiments performed in our laboratory have evaluated the influence of polymerization of diaspirin alpha-alpha cross-linked Hb (alphaalpha-DBBF-Hb) with a bifunctional modified PEG, bis(maleoylglycylamide) PEG (BMAA-PEG), in terms of oxygen carrying capacity, redox properties, hypertensive response, and renal clearance in rats. The data presented in this paper specifically evaluate the influence of BMAA-PEG on alphaalpha-DBBF-Hb (Poly-alphaalpha-DBBF-Hb) to identify molecular weight distribution, protein conformation, and site-specific modification, as well as to provide insight into the previously determined in vitro and in vivo functional and vasoactive characteristics of this HBOC. Chemical analysis performed herein reveals nonspecific modifications induced by BMAA-PEG that result in the full modification of alphaalpha-DBBF-Hb leaving no tetrameric cross-linked starting material in solution. These data are inconsistent with the continuing assumption that molecular size (i.e., 64 kDa) has a direct influence on HBOC-mediated vasoactivity and that other protective strategies should be considered to control blood pressure imbalances. 

Diaspirin cross-linked hemoglobin polymerized with poly; Oxygen carriers; Tetrameric species; Vasoactivity; Amino acids; Blood; Data acquisition; Polyethylene glycols; Polymerization; Proteins; Hemoglobin; 2,3 diphosphoglyceric acid; amino acid derivative; bis(maleoylglycylamide) polyethyleneglycol; diaspirin crosslinked hemoglobin; macrogol; nitric oxide; oxygen; scavenger; unclassified drug; article; blood pressure regulation; blood vessel reactivity; chemical analysis; chemical modification; conjugation; hypertension; kidney clearance; molecular size; oxidation reduction potential; polymerization; protein conformation; protein structure; reproducibility; Aspirin; Chromatography, Gel; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Polyethylene Glycols; Polymers; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization