O$\sb2$ transport was examined by measuring the fractional saturation of concentrated hemoglobin solutions flowing through an artificial capillary (diameter $\approx$ 27 $\mu$m). The measured effects of pH, hemoglobin concentration, O$\sb2$ tension, temperature, and organic phosphate were analyzed by a mathematical model which included the geometry of the capillary, parabolic flow inside the lumen, and cooperative O$\sb2$ binding. Oxygen exchange was limited by diffusion and therefore governed by the magnitude of the O$\sb2$ gradient between the intracapillary fluid phase and the external gas space. In uptake experiments, O$\sb2$ flux was determined primarily by the external O$\sb2$ tension (160 mm Hg) because the internal O$\sb2$ pressure was kept small due to chemical combination with hemoglobin. In release experiments, the external O$\sb2$ tension was maintained at zero, and the transport rate was determined by the intracapillary oxygen partial pressure, which was proportional to the O$\sb2$ half-saturation pressure of the hemoglobin sample. Thus, factors that change the affinity of hemoglobin for oxygen, such as pH, temperature, and organic phosphate concentration, influence strongly the rate of O$\sb2$ release but have little effect on the rate of O$\sb2$ uptake.
The rate-limiting step for erythrocyte CO$\sb2$ transport is the transmembrane exchange of Cl$\sp-$ and HCO$\sb3\sp-$ anions. This process was measured by following extracellular pH changes using a pH-sensitive fluorescent dye in a stopped-flow mixing device equipped with front-face optics. Initial pH and chloride gradients induced pH relaxations which were sensitive to the specific inhibitor 4,4$\prime$-diisothiocyano-2,2$\prime$-stilbenedisulfonic acid (DIDS). Special mixing experiments, designed to minimize anion competition for the transporter binding site(s), were simulated mathematically by models written for ping-pong and random ternary complex mechanisms. The experimentally observed rate dependence on initial extracellular bicarbonate concentration was approximated better by the ping-pong model, and the theoretically derived values for Cl$\sp-$ and HCO$\sb3\sp-$ binding (both K$\sb {\rm D}$'s = 5 mM) as well as the translocation rate (1.1 $\times$ 10$\sp4$ s$\sp{-1}$) agreed very well with literature values for inhibition constants and protein turnover number, respectively. Finally the simultaneous measurement of O$\sb2$ uptake and subsequent HCO$\sb3\sp-$/Cl$\sp-$ exchange was demonstrated. The hemoglobin color change upon oxygen binding caused a rapid change in fluorescence, followed by a slower, DIDS-sensitive pH equilibration.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16255 |
| Date | January 1989 |
| Creators | Lemon, Douglas Dale |
| Contributors | Olson, John S. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 198 p., application/pdf |
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