Interaction of spin-labeled tryptophan with sickle hemoglobin: probing the microenvironment of the contact sites by spin-probe--spin-label techniques.
The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2.10(-5) and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (2H,15N) substituted and deuterated maleimide spin label [2H-15N]MSL covalently-bound to Cys-beta 93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed.
Zeidan
H M
HM
Atlanta University, Chemistry Department, GA 30314.
D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D006451:Hemoglobin, Sickle / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D013113:Spin Labels; D014364:Tryptophan
D006451:Hemoglobin, Sickle; D013113:Spin Labels; D014364:Tryptophan; D003545:Cysteine