Conjoined hemoglobins. Loss of cooperativity and protein-protein interactions.
Hemoglobin cross-linked as a bis(isophthalamide) of the epsilon-amino groups of lysine 82 of each beta-subunit binds and releases oxygen with a Hill coefficient indicative of cooperative oxygen binding (typically approximately 2.0). However, connecting two such cross-linked tetramers with a relatively short covalent linkage produces cross-linked bis-tetramers that bind oxygen with Hill coefficients near unity. To separate the effect of the linkages from the effects of protein-protein interactions in the conjoined proteins, reagents (1 and 2) were used to produce bis-tetramers (A and B). These have a considerably greater distance between cross-linked tetramers than earlier examples. Yet, the bis-tetramers (A and B) bind oxygen with minimal cooperativity (n(50) = 1.4, 1.2). To assess the effect of the linkage itself, cross-linked tetramers (Cand D) were prepared from reactions with the same reagents. These bind oxygen with cooperativity similar to that of cross-linked tetramers without the extended chain (C, n(50) = 2.0; D, n(50) = 1.8). Other tetramers (E and F) with flexible, saturated hydrocarbon appendages were also prepared. These also showed cooperativity in oxygen binding (E, n(50) = 1.7; F, n(50) = 1.8) despite their high degree of hydrophobicity. Thus, the intertetrameric linkages themselves do not induce the loss of cooperativity, leading to the conclusion that solution effects of the tetramers upon one another are the source of the decline in cooperativity: protein-protein interactions are most significant in disrupting the cooperativity of the bis-tetramers, regardless of the span or composition of the linker. This suggests that effects of oligomerization of hemoglobin within red cell substitutes should be considered in terms of such interactions.
Gourianov
Nikolai
N
Davenport Laboratories, Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
Kluger
Ronald
R
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003432:Cross-Linking Reagents; D006454:Hemoglobins; D010100:Oxygen