Biophysical Chemistry 24 (1986) 337-356 Elsevier 337 BPC 01084 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA VISCOSITY OF CONCENTRATED SOLUTIONS AND OF HUMAN ERYTHROCYTE CYTOPLASM DETERMINED FROM NMR MEASUREMENT OF MOLECULAR CORRELATION TIMES zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA THE DEPENDENCE OF VISCOSITY ON CELL VOLUME Zolthn Huba ENDRE** and Philip William KUCHEL * Drparlment of Biochemistry, University zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of Sydney, Sydney, NSW 2006, Australia Received 1st May 1986 Accepted 11th June 1986 Key words: Viscosity; “C-NMR; Erythrocyte; Longitudmal relaxation trmq Correlatron time; Cubic closesf pocking of spheres Metabolically active human erythrocytes were incubated with [a-‘3C]glycine which led to the specific enrichment of intracellular glutathione. The cells were then studied using “C-NMR in which the longitudinal relaxation times (T,) and nuclear Overhauser enhancements of the free glycine and glutathione were measured. The T, values of labelled glycine were also determined in various-concentration solutions of bovine serum albumin and glycerol and also of the natural abundance 13C of glycerol in glycerol solutions. From the T, estimates the rotational correlation time ( .rr) was calculated using a formula based on a model of an isotropic spherical rotor or that of a symmetrical elhpsoidal rotor; for glycine the differences in estimates of 7r obtained using the two models were not significant. From the correlation times and by use of the Stokes-Einstein equations viscosity and translational diffusion coefficients were calculated; thus comment can be made on the likelihood of diffusion control of certain enzyme-catalysed reactions in the erythrocyte. Bulk viscosities of the erythrocyte cytoplasm and the above-mentioned solutions were measured usmg Ostwald capillary viscometry. Large differences existed between the latter viscosity estimates and those based upon NMR-T, measurements. We derived an equation from the theory of the viscosity of concentrated solutions which contains two phenomenological interaction parameters, a ‘shape’ factor and a ‘ volume’ factor; it was fitted to data relating to the concentration dependence of viscosity measured by hoth methods. We showed, by using the equation and interaction-parameter estimates for a particular probe molecule in a particular solution, that it was possible to correlate NMR viscosity and bulk viscosity; in other words, given an estimate of the hulk viscosity, it was possible to calculate the NMR ‘micro’ viscosity or vice versa. However, the values of the interaction parameters depend upon the relative sizes of the prahe and solute molecules and must be separately determined for each probe-solute-solvent system. Under various conditions of extracellular osmotic zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI pressure, erythrocytes change volume and thus the viscosity of the intracellular milieu is altered. The volume changes resulted in changes in the T, of [ a-l3 Clglycine. Conversely, we showed that alterations in r,, when appropriately calibrated, could be used for monitoring changes in volume of metabolically active cells. * To whom correspondence should be addressed. 1. Introduction ** Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. Abbreviations: BSA, bovine serum albumin; Hb, hemoglobin; 9. volume fraction; I(, or, empirical hydrodynamic interaction parameters; T. viscosity: rO, Stokes radius; TV. rotational corre- lation time; T,, longitudinal relaxation time; 6, coefficient of microfriction; U, partial specific volume. Separately, and together, the concentrations of substrates [2] and the intracellular viscosity [3] may influence the rates of metabolic reactions that are necessary for normal cell function. Since erythrocyte shape and rigidity are also influenced by the cell volume and intracellular viscosity, the 0301-4622/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)