Bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate: synthesis, characterization, and reaction with the anion-exchange channel in intact human erythrocytes.
We have synthesized and characterized bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA), a membrane-impermeant bifunctional spin-labeling reagent. BSSDA is a nine carbon backbone homologue of bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate [BSSDP; Beth et al. (1986) Biochemistry 25, 3824-3832]. Due to its longer backbone, BSSDA can span longer distances between reactive groups on a protein than can BSSDP. However, the purpose of the bifunctional design of these reagents is to provide a tight motional coupling of the spin labels to the surface of a target protein. To test whether the longer backbone of BSSDA results in a greater local flexibility and thereby undermines the effects of bidentate attachment, we have labeled with BSSDA anion-exchange channels of intact human erythrocytes at the same site as we have previously labeled them with BSSDP. Linear and saturation-transfer EPR spectra of BSSDA-labeled anion-exchange channels in intact cells closely approximate the corresponding spectra from BSSDP-labeled channels. Thus, the longer backbone of BSSDA relative to BSSDP does not give rise to significant local flexibility, even when BSSDA is bound to a site that can be spanned by the shorter reagent.
Anjaneyulu
P S
PS
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Beth
A H
AH
Cobb
C E
CE
Juliao
S F
SF
Sweetman
B J
BJ
Staros
J V
JV
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000838:Anions; D003432:Cross-Linking Reagents / Q000138:chemical synthesis; D004578:Electron Spin Resonance Spectroscopy; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000378:metabolism*; D007474:Ion Exchange; D010080:Oxazoles / Q000138:chemical synthesis* / Q000378:metabolism; D013113:Spin Labels / Q000138:chemical synthesis; D013388:Succinimides / Q000138:chemical synthesis* / Q000378:metabolism
D000838:Anions; D003432:Cross-Linking Reagents; D007473:Ion Channels; D010080:Oxazoles; D013113:Spin Labels; D013388:Succinimides; C049668:bis(sulfo-N-succinimidyl)doxyl-2-spiro-4'-pimelate; C061440:bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid