Interaction of hemoglobin with the red blood cell membrane. A saturation transfer electron paramagnetic resonance study.
Human hemoglobin has been labeled on cysteine 93(beta) with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and reassociated with erythrocyte membrane previously stripped of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase. The affinity of hemoglobin for the membrane is not affected by the presence of the label. Saturation transfer electron paramagnetic resonance measurements show that the diffusion rotational movements of hemoglobin are considerably slowed down when it is bound to the erythrocyte membrane. The correlation time of rotation, tau c, is found to be 8 . 10(-6) s as compared with 2 . 10(-8) s when the hemoglobin molecule is in solution. The same values are obtained whether the protein is associated with its high- or low-affinity binding sites. They depend on the viscosity of the solution. The high-affinity sites are presumably located on the segment of the band 3 protein which extends into the cytoplasm and which links through ankyrin, the spectrin-actin cytoskeleton to the membrane. When band 3 is cross-linked into a dimer after reaction with the copper-ortho-phenanthroline chelate, the correlation time of rotation of spin-labelled hemoglobin is unchanged. It is also independent of the presence of the spectrin-actin network and ankyrin. These results show tha the movements of hemoglobin bound by ionic linkage to different part (protein or phospholipid) of the cytoplasmic surface of the membrane are similarly highly restricted by some potential or energetic barrier. They give also evidence for independent movements and flexibility in the assembly of the macromolecules which link the spectrin-actin cytoskeleton to the erythrocyte membrane.
D003497:Cyclic N-Oxides / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000097:blood; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000097:blood; D013113:Spin Labels / Q000494:pharmacology
D003497:Cyclic N-Oxides; D006454:Hemoglobins; D008565:Membrane Proteins; D013113:Spin Labels; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases