Transient dichroism studies of spectrin rotational diffusion in solution and bound to erythrocyte membranes.
Spectrin was purified from human erythrocytes and labeled with the triplet probe eosin-5-maleimide. Rotational diffusion of spectrin was investigated by observing transient dichroism following flash excitation of the probe. Measurements were performed at 4 degrees C in solutions of varying viscosity and with spectrin rebound to spectrin/actin-depleted erythrocyte membranes. In solution, complex anisotropy decays were observed which could not be satisfactorily fitted by the equations for a rod-shaped molecule of appropriate dimensions. When spectrin was rebound to the erythrocyte membrane, a decay in the anisotropy was still present but was markedly less sensitive to solution viscosity and flatter at longer times. In order to overcome the objection that the cytoskeleton is only partially reconstituted when spectrin is rebound, a method was developed for labeling spectrin with eosin-5-maleimide in situ. Anisotropy decays for these labeled membranes exhibited features similar to those obtained for spectrin labeled in solution and subsequently rebound. Taken together, the results provide good evidence for segmental motion of spectrin when incorporated into the erythrocyte cytoskeleton. Upon increasing the temperature, the initial anisotropy ro for both rebound and in situ labeled spectrin decreases, and above 30 degrees C the measured anisotropies are small. Thus, at physiological temperature the probe is almost completely randomized by motions with correlation times less than 10 microseconds.
Department of Chemistry and Biological Chemistry, University of Essex, Colchester, U.K.
M J
MJ
Clague
J P
JP
Harrison
I E
IE
Morrison
K
K
Wyatt
R J
RJ
Cherry
D002942:Circular Dichroism; D004058:Diffusion; D004801:Eosine Yellowish-(YS); D004910:Erythrocyte Membrane / Q000032:analysis*; D006801:Humans; D012996:Solutions; D013049:Spectrin / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D012996:Solutions; D013049:Spectrin; D004801:Eosine Yellowish-(YS)