From monitoring of the exchangeable proton resonance at 14 ppm and the hyperfine-shifted proton resonances of the cyanomet chain of these hybrid Hbs in the deoxy and carbonmonoxy forms, the following conclusions have been reached (i) the T-quaternary structureofthetwosinglyligatedspeciesinthedeoxy form is altered as manifested by a reduction of the intensity of the T-structural marker at 14 ppm, compared to that of deoxy-Hb A; (ii) hybrid Hbs with two cyanomet chains per Hb tetramer have no observable exchangeable proton resonance at 14 ppm, suggesting that they are not in the T-quaternary state; and (iii) the structural changes of these crosslinked mixed-valence hybrid Hbs as a function of ligation are not concerted and cannot be explained simply by two-structure concerted models (Miura and Ho, 1982).
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