Major Research Interests

Our laboratory has been using biochemical, biophysical, chemical, and chemical engineering approaches to examine the fundamental processes involved in oxygen transport and storage in mammalian circulatory systems. We are directing two major research programs, one involving basic molecular biophysics and the other involving the design of O2 delivery pharmaceuticals.

(1) Dynamics of O₂ binding to heme proteins and reactions with NO - Mammalian myoglobin and the subunits of human hemoglobin are being used as simple prototypes. The roles of specific amino acids in regulating the kinetics and affinity of O₂ binding and the resistance of the protein to unfolding are being identified by a wide variety of biophysical, structural, and computational methods. The mechanisms derived to explain regulation of O₂ affinity and kinetics and inhibition of oxidative side reactions in myoglobins and hemoglobins are applicable to all heme proteins, from cytochrome c oxidase, the key enzyme in respiration, to the P450 oxygenases involved in detoxification of xenobiotics. Our library of ~350 rationally designed myoglobin mutants are also being used to study the reaction of NO with bound O₂. This reaction results in the formation of a transient Fe(III)-peroxynitrite intermediate that rapidly isomerizes into nitrate and free metmyoglobin. Finally, recombinant myoglobins and hemoglobins are excellent model systems for understanding the structural compromises between protein stability and physiological function that occur during evolution.

(2) The design of extracellular hemoglobin-based blood substitutes - Our molecular biophysical studies have provided specific strategies for designing more efficient and safe O₂ delivery pharmaceuticals. Key properties of an efficient and economical hemoglobin-based blood substitute are moderate O₂ affinity, large O₂ dissociation rate constants, high cooperativity, resistance to denaturation, and high expression yields in E. coli. The mechanisms and methodologies derived from our biophysical projects provide empirical and theoretical frameworks for optimizing these properties. Our in vitro studies of the reactions of NO with oxygenated heme proteins served as the scientific framework for determining the cause of blood pressure elevation by extracellular hemoglobin and for developing a strategy to eliminate this side effect. These ideas and interpretations were used by Baxter Hemoglobin Therapeutics, Boulder, CO (formerly Somatogen, Inc.) to construct more efficient and safer second-generation blood substitutes and resulted in three joint patents. Unfortunately, Baxter International dropped its recombinant Hb-based blood substitute project in July of 2003, presumably because of production problems. We are currently working on ways to increase the expression of rHb in E. coli using wide variety of genetic engineering approaches.