Effects of beta 6 aromatic amino acids on polymerization and solubility of recombinant hemoglobins made in yeast.
Valine, leucine, tryptophan, and phenylalanine substitutions at the beta 6 position of hemoglobin (Hb) were made using a yeast expression system coupled with a polymerase chain reaction-based mutagenesis strategy. The oxygen affinity and absorption spectra of these mutants were similar to recombinant Hb A except for Hb beta E6W which had a higher absorbance at approximately 280 nm. The deoxy forms of Hb beta E6L and Hb S showed characteristic delay times prior to polymerization. Tetrameric deoxy-Hbs containing tryptophan or phenylalanine at the beta 6 position had higher solubilities and polymerized less readily compared with deoxy-Hb S. However, when oversaturated, these Hbs polymerized without a delay time. These results suggest that Hb beta E6W and Hb beta E6F form polymers upon deoxygenation by a linear polymerization mechanism without nuclei formation. During polymerization, bulky hydrophobic amino acids, like phenylalanine and tryptophan at the beta 6 position, might interact with the acceptor pocket on the surface of an adjacent Hb molecule but may not be able to form stable hydrophobic interactions like beta 6 valine and leucine. Difficulty in insertion of the bulky side chains of these aromatic amino acids into the hydrophobic acceptor pocket on an adjacent tetramer may inhibit nuclei formation prior to polymerization.
Adachi
K
K
Children's Hospital of Philadelphia, Department of Pediatrics, Pennsylvania 19104.
Konitzer
P
P
Kim
J
J
Welch
N
N
Surrey
S
S
D000596:Amino Acids / Q000737:chemistry*; D004588:Electrophoresis, Cellulose Acetate; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D016296:Mutagenesis; D011108:Polymers; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D012995:Solubility; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D006454:Hemoglobins; D011108:Polymers; D011994:Recombinant Proteins