4-maleimido-2-2-6-6-tetramethylpiperidinooxyl and Anemia--Sickle-Cell

The spin-labeled tryptophan (TrpSL) was used as a structural probe of hemoglobin (Hb) contact sites. The electron paramagnetic resonance spectral data indicated that the probe exhibits weak binding to Hb with a dissociation constant of 3.2 x 10(-5) and 4.0 mol bound per Hb tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.5 s. The topology of the contact sites was investigated by using a dual spin label methodology in which TrpSL and 2H-15N covalently bound to B 93 cysteine residue were used. The electron spin resonance spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled Hb. The environment of the contact sites is discussed.

Topics: Anemia, Sickle Cell; Binding Sites; Cyclic N-Oxides; Cysteine; Electron Spin Resonance Spectroscopy; Hemoglobin, Sickle; Hemoglobins; Humans; Spin Labels; Tryptophan