Intermediates in the interconversion of acid and alkaline ferrihemoglobin. Structural and kinetic aspects.

The acid-alkaline pH-jump in suspension of crystalline sheep hemoglobin has been studied in the range of 5.95 to 8.94. Crystals suspended in 3.8 M Cs2SO4 show a rapid optical transition of half-time equal to or less than 2 ms. As the ammonia concentration is increased in the Cs2SO4-suspended crystals, a second optical transition is observed as a pseudo-first-order reaction, with a rate constant of between 10 and 15 s-1. The alkaline-acid pH-jump proceeds through a very rapid shift of the alkaline-acid equilibrium and is followed by a first-order dissociation constant between 9 and 12 s-1. The dissociation of the ammonia is biphasic, and the ratio between the fast and slow phases is 9.

The reaction kinetics of fluoride and ammonia with acid and alkaline ferrimyoglobin in a crystalline state.

Differences between the reactivity of amorphous and crystalline myoglobin have been studied by the rapid-flow method combined with dual-wavelength spectrophotometry. The binding of ammonia to the hydroxide compound has a half-time of 55 ms. The reverse reaction has a half-time of 70 ms. At pH 7.0 the relative half-times of combination and dissociation with fluoride are 10 min for crystalline and 1.8 min for amorphous materials. Reactivity of the crystals to fluoride at pH 6.0 greatly increased as compared with pH 8.7. Half-time at pH 8.7 is 10 min, while at pH 6.0 the half-time is 2.5 s for the crystalline material and 1.4 s for the amorphous material. The exchange of fluoride by azide at pH 6.0 is 3.1-fold faster in amorphous material than in crystalline material.

The binding of asparagine, glutamine and homoserine to human erythrocytes containing hemoglobins S and CS and to soluble hemoglobins S and CS.

Tritium labeled asparagine binds to oxyhemoglobin S and to a mixture of hemoglobins C and S in the molar ratio of 3.38:1 and 8.2:1 respectively. From the dialysis equilibrium studies it appears that labeled asparagine does not bind to oxy- or deoxy- hemoglobin A nor to deoxyhemoglobin S. The constant for equilibrium association of asparagine for oxyhemoglobin S is 7.38 x 10(7) M(-1) and for oxyhemoglobin CS 4.8 X 10(4) M(-1) at 23 degrees C. Tritium labeled asparagine is bound to oxyhemoglobin S and CS sufficiently strongly to prevent dissociation under the conditions of gel electrophoresis at pH 9.50. The protein with and without bound asparagine, glutamine or homoserine, is indistinguishable in molecular net charge and size by the criteria of quantitative polyacrylamide gel electrophoresis (PAGE). Also there were no significant differences in mobility between hemoglobin S and hemoglobin C in the presence and absence of asparagine, glutamine and homoserine as detectable in agar coated cellulose acetate electrophoresis at pH 6.3.

Inhibition of sickling in erythrocytes by amino acids.

Sickling of erythrocytes was rapidly and quantitatively inhibited at room temperature by 3.8 mM homoserine, asparagine, and glutamine but by no other amino acid.