DO THE SPECTRA OF MALEIMIDE SPIN-LABELED WHOLE-BLOOD PLATELETS REFLECT THE STRUCTURE AND CONFORMATION OF MEMBRANE-PROTEINS

The maleimide spin label (4-maleimido-2,2,6,6-tetramethylpiperidine-1- oxyl, MSL), the commonly used reagent specific for cysteine thiol grou ps in proteins, penetrates cell membranes and binds to both the membra ne and cytoplasmic protein moieties. In order to differentiate the lab elling of these two subpopulations of cell proteins, we developed thre e different methodological approaches varying in the consequent distri bution of this label in platelets. (A) The labelling of platelet prote ins was negligible when bovine serum albumin was used in the Tyrode's buffer for the isolation of platelets, as the majority of the spin lab el was bound to the albumin coated on the platelets. (B) Preblocking o f the reactive thiol groups in albumin with non-spin maleimide analog, N-ethylmaleimide (NEM), caused a considerable amount of MSL to bind w ith whole platelets but the impartment of membrane component was below 50%. It suggests that the majority of the spin label penetrated plate lets and was bound to the intrinsic platelet proteins. (C) In order to prevent labelling of intrinsic platelet proteins with MSL, platelets were preincubated with N-ethylmaleimide, which was able to penetrate p latelets and block the reactive thiol groups inside the cells. Such a treatment resulted in a saturation of the intrinsic protein residues w ith this non-spin analog. The subsequent incubation of thus-treated al bumin-free platelets with MSL was to enhance considerably the likeliho od of the attachment of MSL molecules to the thiol groups available in platelet-membrane proteins. Indeed, the incubation of thus-prepared p latelets favoured the binding of MSL to the thiol groups of platelet-m embrane proteins but not completely, since over 35% of MSL penetrated platelets, and consequently was bound also to intrinsic proteins. In a relatively wide range of MSL concentrations it was impossible to satu rate membrane proteins without simultaneous labelling of platelet intr insic proteins to a considerable extent. Our results evidently indicat e that the capacity for the spin label inside platelets exceeds consid erably that of platelet membranes. Thus, the isolation of a particulat e platelet membrane fraction is the only procedure which allows the la belling of platelet membrane proteins selectively.