A light scattering investigation of the propylurea dissociation of human hemoglobin A.
The subunit dissociation of human hemoglobin A by propylurea in several liganded and chemically modified states was investigated by light scattering molecular weight methods. The dissociation data were analyzed by means of the equation developed in our earlier studies: deltaF degrees D EQUALS TO DELTAF degrees D, W - 2N'RTKB[D], where deltaF degrees D and deltaf degreees D,W represent the free energy of dissociation of hemoglobin tetramers into half-molecules consisting of alpha beta dimers in the presence and in the absence of propylurea, KB is the binding constant of the urea to the average peptide unit, [D] is its concentration, and N' is the number of amino acid sites exposed per half-molecule on dissociation. It is found that the dissociation of oxyhemoglobin, cyanmethemoglobin, and N-ethylmaleimide oxyhemoglobin is characterized by essentially the same N' value of 15 to 21 plus or minus 3, that are close to the 19 amino acid residues per surface which comprise the smaller alpha beta contact area, seen in the X-ray crystallographic model of horse hemoglobin of Perutz and coworkers. Due to the very low degree of dissociation of deoxyhemoglobin, only a very approximate estimate of N' of about the same order of magnitude could be obtained for this form of the protein. In contrast, a significantly lower value of N' was obtained with bis(maleimidomethyl) ether modified oxyhemoglobin of 8 plus or minus 3, that is cross-linked at cysteine residue F9 (93)beta and histidine residue FG4 (97) beta in the same beta chains. Our results suggest that alterations caused by the presence of the cross-linking reagent reflect both the loss in amino acid residues that can interact with the urea at the blocked segments of the polypeptide chains in the dissociated state of hemoglobin and the changes in accessibility of some of the amino acid residues perturbed by the introduction of the reagent in the parent tetrameric form.
D000433:1-Propanol; D000328:Adult; D001665:Binding Sites; D003486:Cyanides; D005033:Ethylmaleimide; D006454:Hemoglobins; D006801:Humans; D008024:Ligands; D008027:Light; D046911:Macromolecular Substances; D008433:Mathematics; D008970:Molecular Weight; D010108:Oxyhemoglobins; D011485:Protein Binding; D012542:Scattering, Radiation; D014508:Urea
D003486:Cyanides; D006454:Hemoglobins; D008024:Ligands; D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D014508:Urea; D000433:1-Propanol; D005033:Ethylmaleimide