Proton nuclear magnetic resonance investigation of cross-linked asymmetrically modified hemoglobins: influence of the salt bridges on tertiary and quaternary structures of hemoglobin.

Asymmetrically modified hemoglobins, [alpha(des-Arg)beta]A[alpha beta]cXL, [alpha(des-Arg-Tyr)beta]A[alpha beta]cXL, [alpha(des-Arg)beta(NES)]A[alpha beta]cXL, and [alpha(des-Arg)beta]A[alpha beta(NES)]cXL, have been prepared from chemically modified human normal adult hemoglobin (Hb A) and mutant hemoglobin C (beta 6Glu----Lys), where the subscript A or C denotes that the alpha beta dimer is from either Hb A or Hb C, respectively, and XL symbolizes a cross-linked hemoglobin prepared by reaction with a bifunctional cross-linking reagent, bis(3,5-dibromosalicyl) fumarate. It has been shown by X-ray crystallography that this bifunctional reagent cross-links the epsilon-amino group of the lysyl residue at position 82 of the two beta chains [Walder, J. A., Walder, R. Y., & Arnone, A. (1980) J. Mol. Biol. 141, 195]. Proton nuclear magnetic resonance spectra of these asymmetrically modified hemoglobins together with their parent hemoglobins, des-Arg(alpha 141) Hb A, des-Arg(alpha 141)-Tyr(alpha 140) Hb A, NES-Hb A and NES-des-Arg(alpha 141) Hb A, have been obtained over the spectral region 5-10 ppm downfield from H2O for the exchangeable proton resonances and 50-80 ppm downfield from H2O for the hyperfine-shifted proximal histidyl N delta H exchangeable proton resonances. The experimental results indicate that the effects on the hyperfine-shifted proximal histidyl N delta H exchangeable proton resonances at pH 6.0 of removing Arg(alpha 141) or Arg(alpha 141)-Tyr(alpha 140) from one of the two alpha subunits are limited to within the alpha subunit from which the carboxyl-terminal amino acids are specifically removed.(ABSTRACT TRUNCATED AT 250 WORDS)