The βLys66Tyr Variant of Human Hemoglobin as a Component of a Blood Substitute.

• It has been proposed that introducing tyrosine residues into human hemoglobin (e.g. βPhe41Tyr) may be able to reduce the toxicity of the ferryl heme species in extracellular hemoglobin-based oxygen carriers (HBOC) by facilitating long-range electron transfer from endogenous and exogenous antioxidants. Surface-exposed residues lying close to the solvent exposed heme edge may be good candidates for mutations. We therefore studied the properties of the βLys66Tyr mutation. Hydrogen peroxide (H2O2) was added to generate the ferryl protein. The ferryl state in βLys66Tyr was more rapidly reduced to ferric (met) by ascorbate than recombinant wild type ((r)wt) or βPhe41Tyr. However, βLys66Tyr suffered more heme and globin damage following H2O2 addition as measured by UV/visible spectroscopy and HPLC analysis. βLys66Tyr differed notably from the (r)wt protein in other ways. In the ferrous state the βLys66Tyr forms oxy, CO, and NO bound heme complexes similar to (r)wt. However, the kinetics of CO binding to the mutant was faster than (r)wt, suggesting a more open heme crevice. In the ferric (met) form the typical met Hb acid-alkaline transition (H2O to (-)OH) appeared absent in the mutant protein. A biphasicity of cyanide binding was also evident. Expression in E. coli of the βLys66Tyr mutant was lower than the (r)wt protein, and purification included significant protein heterogeneity. Whilst, βLys66Tyr and (r)wt autoxidised (oxy to met) at similar rates, the oxygen p50 for βLys66Tyr was very low. Therefore, despite the apparent introduction of a new electron transfer pathway in the βLys66Tyr mutant, the heterogeneity, and susceptibility to oxidative damage argue against this mutant as a suitable starting material for a HBOC.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Läkemedelskemi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Medicinal Chemistry (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

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