Use of sensitized fluorescence for the study of the exchange of subunits in protein aggregates.
The exchange of subunits between oligomer protein particles depends upon a cycle of dissociations and associations. To examine the dynamics of these cycles we have employed two methods based on the transfer of excitation energy between fluorochromes attached to different subunits of protein oligomers, at various temperatures and pressures. In the heterotransfer method, identical solutions independently labeled with two different fluorophores, donor D and acceptor A, are mixed. The fluorescence spectrum permits the determination of the subunit exchange by the increase in A and decrease in D fluorescence as mixed AD oligomers are formed. In the homotransfer method the aggregates are labeled with fluorescein to the extent that, ideally, each subunit carries a fluorophore. The emission is strongly depolarized because sufficiently often it takes place after a transfer to a fluorophore oriented differently from the one originally excited. Both dissociation and subunit exchange with unlabeled material result in an increase in polarization and can be independently determined by the homotransfer method. Both homo- and heterotransfer have been employed in the study of the effect of temperature on the stability of the aggregates and the relation between the rate of dissociation and the rate of exchange when dissociation of oligomers is induced by hydrostatic pressure.
Erijman
L
L
Department of Biochemistry, School of Chemical Science, University of Illinois at Urbana-Champaign 61801.
Weber
G
G
D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry*; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine; D013816:Thermodynamics; D013997:Time Factors
D007527:Isoenzymes; D046911:Macromolecular Substances; D011506:Proteins; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases