Anomalous pressure dissociation of large protein aggregates. Lack of concentration dependence and irreversibility at extreme degrees of dissociation of extracellular hemoglobin.
The effects of hydrostatic pressure on the extracellular hemoglobin of Glossoscolex paulistus were investigated by studies of light scattering, intrinsic protein fluorescence, filtration chromatography, and oxygen binding. Pressure promoted a large decrease of light scattering consistent with the dissociation of the hemoglobin. Pressures up to 1.7 kbar caused dissociation with reversibility of the light scattering and fluorescence properties after return to atmospheric pressure. Higher pressures provoked additional dissociation with increasing loss of reversibility. After complete dissociation by incubation at 2.5 kbar followed by decompression, the protein continued mostly dissociated. The dissociated forms were distributed in two populations as based on size exclusion chromatography, one corresponding to small dissociated units (average Mr = 33,000) and the other population corresponding to the one-twelfth subunit (260,000 Mr). The pressure dissociation curves showed no significant dependence on protein concentration suggesting that the native hemoglobin population exists in a distribution of free-energies of association. Both the decrease of concentration dependence and the loss of ability to reassemble seem to increase with the complexity and size of the protein aggregate. These findings permit the conclusion that increased heterogeneity of free-energies of association with the size of the aggregate may result in the molecular individuality of large protein complexes such as subcellular particles and viruses.
Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Brazil.
J L
JL
Silva
M
M
Villas-Boas
C F
CF
Bonafe
N C
NC
Meirelles
D000818:Animals; D000845:Annelida; D002851:Chromatography, High Pressure Liquid; D006454:Hemoglobins / Q000378:metabolism*; D006874:Hydrostatic Pressure; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008970:Molecular Weight; D010108:Oxyhemoglobins / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
D006454:Hemoglobins; D046911:Macromolecular Substances; D010108:Oxyhemoglobins