Do the spectra of maleimide spin-labelled 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 groups in proteins, penetrates cell 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Watala
C
C
Department of Biophysics, Medical University of Lodz, Poland.
Pietrucha
T
T
Gwoździński
K
K
Cierniewski
C S
CS
D000818:Animals; D001792:Blood Platelets / Q000737:chemistry*; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry*; D011487:Protein Conformation; D013113:Spin Labels; D013552:Swine
D003497:Cyclic N-Oxides; D008565:Membrane Proteins; D013113:Spin Labels; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl