Enolase Dissociation Induced by High Pressure. A Study by Fluorescence Polarization
Paladini, Alejandro Alberto
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Permalink
https://hdl.handle.net/2142/67595
Description
Title
Enolase Dissociation Induced by High Pressure. A Study by Fluorescence Polarization
Author(s)
Paladini, Alejandro Alberto
Issue Date
1980
Department of Study
Physiology and Biophysics
Discipline
Biophysics
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
Language
eng
Abstract
Enolase is a protein of M.W. 90 kilodaltons, formed by two identical subunits of 45 kilodaltons each. Fluorescence polarization is the main spectroscopic property followed to characterize the dissociation of this enzyme under pressure.
In order to be able to measure this property under high pressure a cell was specially designed and constructed. The birefringency of the cell windows was determined to permit measurement of the true polarization changes of the biological system.
Experimental data using polarization of the intrinsic ultra violet fluorescence of the protein shows for enolase a change in volume upon dissociation of about -70 ml/mole. The free energy of dissociation measured at atmospheric pressure was about 9 kcal mole('-1), a value in close agreement with the literature. The polarization of the ultra violet fluorescence reflects mainly the increased freedom of rotation of tryptophan residues on dissociation.
Enolase was also labeled with dansylchloride and the polarization under pressure obtained. The values of the polarization in this case reflect mainly the volume of the particles.
Reversibility of the system measured in terms of polarization reproducibility after each pressure run is better than 98%. Theoretical analysis of the experimental data also allowed us to determine the decrease in volume upon dissociation at 1 atmosphere (-18 ml mole('-1)) and the compressibility of the residues at the boundary between the subunits (-36 ml mole('-1) kbar('-1)).
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