PROTEIN ENGINEERING STRATEGIES FOR DESIGNING MORE STABLE HEMOGLOBIN-BASED BLOOD SUBSTITUTES

Over the past five years our laboratory has been using rational, compa rative, and random combinatorial mutagenesis strategies to optimize th e alpha and beta subunits of recombinant human hemoglobin (Hb) for eff icient O-2 transport, greater stability, and minimum interference with vascular activity. In each approach, mammalian myoglobin (Mb) has bee n used as a prototype to develop experimental methodologies and to stu dy the stereochemical mechanisms that govern O-2 affinity, discriminat ion against CO, rates of ligand binding, auto- and chemically induced oxidation, resistance to hemin loss, and stability to globin denaturat ion. Multiple replacements in the distal portion of the heme pocket ha ve been designed rationally to lower oxygen affinity and at the same t ime inhibit oxidative side reactions. The P-50 values are adjusted by altering electrostatic and steric interactions between the bound ligan d and residues at the Leu(B10), His(E7), and Val(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 exclu ding oxidants and proton donors from the immediate vicinity of the bou nd ligand. Similar strategies appear to have evolved in a number of an imal myoglobins and hemoglobins which have unusual amino acids at the E7, B10, and E11 positions. Random combinatorial mutagenesis technique s have been developed to insert new amino acid combinations near the b ound ligand in sperm whale Mb. The objective is to obtain ''unnatural' ' distal pocket structures that enhance O-2 transport and resistance t o oxidation by alternative mechanisms.