The kinetics of ligand binding for diverse mammalian myoglobins and the effects of substitutions outside the heme cavity.

1. We report rate constants for oxygen dissociation and for oxygen, carbon monoxide, azide, and cyanide binding to whale, horse, dog, beef and human myoglobins. 2. For azide binding, rate constants can vary by at least a factor of two for substitutions outside the heme cavity. Azide binding may be affected by a substitution at residue 66 in the E helix, a site suggested by Case & Karplus (1979) J. molec. Biol. 132, 343-368, to be on a reactive path to the heme. 3. The oxygen and CO data show that substitutions outside the heme cavity can affect rate constants by at least a factor of 1.5. 4. The oxygen equilibrium constant was correlated with the metabolite rate of the corresponding species, in accord with the Wyman (1966) J. biol. Chem. 241, 115-121, model for facilitated diffusion of oxygen.

Glycine transport by hemolysed and restored pigeon red cells. Effects of a Donnan-induced electrical potential on entry and exit kinetics.

The influence of a Donnan effect on the transport of glycine by hemolysed and restored pigeon red cells was examined. The Donnan effect was produced by replacing Cl- with 2,4-toluenedisulfonate or glutamate. The effects of the associated membrane potential and inside-outside pH difference on glycine entry and exit rates were examined. The effects of pH on entry and exit rates in the absence of a Donnan effect were also examined. In the absence of a Donnan effect, Na+-dependent glycine entry requires the protonated form of a group with a pKapp of 7.9 and the deprotonated form of another group with a pKapp of 6.8. Neither of these are required for exit but the deprotonated form of a group(s) with a pKapp of 6.2 is required. The pK 7.9 group and pK 6.2 group probably react with H+ at the inner face of the membrane and the pK 6.8 group probably reacts at the outer face. The V for glycine entry was determined for cells with their Cl- largely replaced by toluenedisulfonate and without such replacement. Between pH 6.1 and 7, the ratio of the respective V values, VT/VC1, was 1.5-1.7. VT/VC1 rose above pH 7 to near 4 at pH 8.3. At pH 6.9, with glutamate replacing cell Cl-, the analogous ratio (VGlu/VC1) was 1.7. The increase of VT/VC1 above pH 7 could be quantitatively accounted for by the increase in cell [H+]/medium [H+] caused by the Donnan effect together with the assumption that the pK 7.9 group reacts with H+ at the inner face of the membrane. When cell Cl- was replaced by toluenedisulfonate or glutamate there was a drop in the term in the glycine Km describing Na+ dependence of glycine entry. When cell Cl- was replaced by toluenedisulfonate therewas a rise in the Na+-independent term in the glycine entry Km. By replacing varying amounts of cell Cl- with either toluenedisulfonate or glutamate, plots were obtained of entry rates vs. the cell [Cl-]/ medium [Cl-] ratio consistent with the assumption that the Donnan-induced membrane potential acts on a "moving" charge. Glycine exit was only slightly accelerated by trans-toluenedisulfonate. The ratio, exit rate into toluenedisulfonate medium/exit rate into Cl- medium rose with decreasing pH. This rise could be accounted for by a Donnan-induced inside-outside pH difference which affects a pKapp 6.2 group reacting with internal H+. The observed influences of the Donnan effect on V (glycine entry), on both components of Km (glycine entry), on the shape of the plot of glycine entry rate vs. the cell [Cl-]/medium [Cl-] ratio and on glycine exit all fit the assumptions that when the empty porter reorients, one unit of negative charge accompanies it "across" the membrane and that no other steps involve charge movement. The properties of the system seem inconsistent with a translational ("ferry boat") mobile carrier.