title: Protein engineering strategies for designing more stable hemoglobin-based blood substitutes.
abstract: Over the past five years our laboratory has been using rational, comparative, and random combinatorial mutagenesis strategies to optimize the alpha and beta subunits of recombinant human hemoglobin (Hb) for efficient O2 transport, greater stability, and minimum interference with vascular activity. In each approach, mammalian myoglobin (Mb) has been used as a prototype to develop experimental methodologies and to study the stereochemical mechanisms that govern O2 affinity, discrimination against CO, rates of ligand binding, auto- and chemically induced oxidation, resistance to hemin loss, and stability to globin denaturation. Multiple replacements in the distal portion of the heme pocket have been designed rationally to lower oxygen affinity and at the same time inhibit oxidative side reactions. The P50 values are adjusted by altering electrostatic and steric interactions between the bound ligand and residues at the Leu(B10), His(E7), and Va(E11) positions. Large apolar residues (Leu, Phe, Trp) at the B10 and E11 positions inhibit NO-induced and autooxidation in both myoglobin and hemoglobin by excluding oxidants and proton donors from the immediate vicinity of the bound ligand. Similar strategies appear to have evolved in a number of animal myoglobins and hemoglobins which have unusual amino acids at the E7, B10, and E11 positions. Random combinatorial mutagenesis techniques have been developed to insert new amino acid combinations near the bound ligand in sperm whale Mb. The objective is to obtain "unnatural" distal pocket structures that enhance O2 transport and resistance to oxidation by alternative mechanisms.
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251-1892, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Olson
authors.forename: R F
authors.initials: RF
authors.lastname: Eich
authors.forename: L P
authors.initials: LP
authors.lastname: Smith
authors.forename: J J
authors.initials: JJ
authors.lastname: Warren
authors.forename: B C
authors.initials: BC
authors.lastname: Knowles
mesh_terms: D001802:Blood Substitutes / Q000138:chemical synthesis*; D006454:Hemoglobins / Q000138:chemical synthesis* / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins