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Do the spectra of maleimide spin-labelled whole blood platelets reflect the structure and conformation of membrane proteins?

Authors
Journal
Journal of Biochemical and Biophysical Methods
0165-022X
Publisher
Elsevier
Publication Date
Volume
27
Issue
2
Identifiers
DOI: 10.1016/0165-022x(93)90060-2
Keywords
  • Platelet Membrane
  • Membrane Protein
  • Epr Spectroscopy
  • Spin Label
  • Maleimide Spin Label
  • (Porcine Platelet)
Disciplines
  • Biology
  • Mathematics

Abstract

Abstract The maleimide spin label (4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl, MSL), the commonly used reagent specific for cysteine thiol groups in proteins, penetrates cells membranes and binds to both the membrane and cytoplasmic protein moieties. In order to differentiate the labelling of these two subpopulations of cell proteins, we developed three different methodological approaches varying in the consequent distribution of this label in platelets. (A) The labelling of platelet proteins was negligible when bovine serum albumin was used in the Tyrode's buffer for the isolation of platelets, as the majority of the spin label was bound to the albumin coated on the platelets. (B) Preblocking of the reactive thiol groups in albumin with non-spin maleimide analog. N-ethylmaleimide (NEM), caused a considerable amount of MSL to bind with whole platelets but the impartment of membrane component was below 50%. It suggests that the majority of the spin label penetrated platelets 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 platelets and block the reactive thiol groups inside the cells. Such a treatment resulted in a saturation of the intrinsic protein residues with this non-spin analog. The subsequent incubation of thus-treated albumin-free platelets with MSL was to enhance considerably the likelihood of the attachment of MSL molecules to the thiol groups available in platelet-membrane proteins. Indeed, the incubation of thus-prepared platelets favoured the binding of MSL to the thiol groups of platelet-membrane 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 saturate membrane proteins without simultaneous labelling of platelet intrinsic proteins to a considerable extent. Our results evidently indicate that the capacity for the spin label inside platelets exceeds considerably that of platelet membranes. Thus, the isolation of a particulate platelet membrane fraction is the only procedure which allows the labelling of platelet membrane proteins selectively.

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