Nuclear Magnetic Resonance Studies of Biological Systems

Citation

Antypas, William George, Jr. (1988) Nuclear Magnetic Resonance Studies of Biological Systems. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/j4ep-gn92. https://resolver.caltech.edu/CaltechTHESIS:01182013-100334158

Abstract

Chapter 1: pH Homeostasis in Human Red Blood Cells

The intracellular pH of normal and homozygous sickle cell red blood cells was measured in cell suspensions in plasma by NMR. Freshly drawn, metabolically active red cells maintain a transmembrane pH gradient that differs significantly from the expected Donnan equilibrium proton distribution. In the physiologically important extracellular pH range of 7.12 to 7.57 the intracellular pH is maintained within the narrow range of 7.20 to 7.37. Outside of this range, the intracellular pH is linearly dependent on the extracellular pH. Thus, red cells maintain an intracellular pH that is closer to 7.3 over the entire extracellular pH range than is expected from the Donnan equilibrium ion distribution. The ligation state of cellular hemoglobin shifted the position of the intra- vs extra-cellular pH relationship, but did not alter the ability of the cells to regulate intracellular pH. Deoxygenation of normal red blood cells resulted in an intracellular pH increase of 0.05 ± 0.02 compared to oxygenated cells over the extracellular pH range 7.00 to 7.80. Metabolically depleted cells are unable to maintain a non-Donnan equilibrium proton distribution. The regulation of intracellular pH was regained by restoring cellular ATP levels. Sickle cell blood demonstrated the same ability to regulate intracellular pH as was observed in normal blood. Deoxygenation of sickle cell blood also resulted in a net increase in intracellular pH. However, gelation of the sample prevented accurate intracellular pH measurements of completely deoxygenated sickle cell samples.

Chapter 2: Measurements of Cell Volume by Nuclear Magnetic Resonance

The Mean Hemoglobin Concentration (MHC) of red blood cells was measured non-invasively and non-destructively by NMR. The difference between intracellular and extracellular proton relaxation rates provides the basis for the determination of the MHC in red blood cells. T₁ relaxation times were measured at a proton frequency of 200 MHz. The T₁ relaxation time for water protons in serum is 2.20 ± 0.20 seconds. The T₁ relaxation time of water protons in red blood cell pellets is 0.64 ± 0.15 seconds. In red blood cell lysate, the T₁ relaxation time is 0.77 ± 0.11 seconds. The observed water T₁ relaxation data from red blood cell samples under various conditions were fit to the complete equation for the time-dependent decay of magnetization for a two-compartment system including chemical exchange. The MHC for each sample was calculated from the hematocrit and the intracellular water fraction as determined by NMR. MHC values obtained in this manner ranged from 25% to 29% by volume for normal red blood cells in serum, in agreement with published values. The use of proton NMR to determine MHC values directly and non-destructively provides a method to evaluate the effect of various agents on the MHC in viable cells and has wide applicability to the study of antisickling agents in intact cells. The ability to monitor cell volume and to follow the effect of agents known to affect ion transport (valinomycin, nystatin, amiloride, etc.) on cell volume has enormous experimental potential.

Chapter 3: ³¹P NMR Studies of the Binding Site of anti-Phosphorylcholine Antibodies

The binding of the phosphorylcholine (PC) analogue, 2-(trimethylphosphonio)-ethylphosphate (phosphorylphosphocholine, PPC) to the PC binding myeloma proteins TEPC-15, McPC 603, and MOPC 167 was studied by ³¹P NMR. Binding of PPC to each of the proteins results in an observed phosphate chemical shift that is identical to the shift observed when PC is bound. Thus, the specific binding interactions of the phosphate subsite of the proteins with PC are maintained when PPC is bound. The chemical shift and titration behavior of the phosphonium resonance of PPC was studied as a probe of the choline subsite of these proteins. PPC bound to TEPC-15 or MOPC 167 exhibits a +0.1 ppm upfield shift from the free hapten. In contrast, PPC binding to McPC 603 results in a +2.7 ppm upfield shift that titrates with a pK_a of 3.6. The shape of the titration curve indicates that the ionizations of 2 protons of equal pK_a are responsible for the observed titration. Three acidic residues provide the major contribution to the choline subsite in both TEPC-15 and MOPC 167. The amino acid substitution ASP_99H → Asn in the third hypervariable region of McPC 603 destroys the spatial symmetry of the choline subsite in McPC 603. The symmetry of the charge distribution of the choline subsite that is lost by this substitution is restored at low pH by titrating the negative charges of Glu_35H and Glu_59H.

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