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Buffered NaCI solutions hypertonic to rabbit serum were prepared and freezing point depressions of each determined after dilution with measured amounts of water. Freezing point depression of these dilutions was a linear function of the amount of water added. One ml. of rabbit red cells was added to each 4 ml. of the hypertonic solutions and after incubation at 38°C. for 30 minutes the mixture was centrifuged and a freezing point depression determined on the superuatant fluid. The amount of water added to the hypertonic solutions by the red ceUs was calcuated from this freezing point depression. For each decrease in the freezing point of -0.093 °C. of the surrounding solution red cells gave up approximately 5 ml. of water per 100 ml. of red cells in the range of --0.560 to -0.930°C. Beyond -0.930°C. the amount of water given up by 100 ml. of red cells fits best a parabolic equation. The maximum of this equation occurred at a freezing point of the hypertonic solution of --2.001°C. at which time the maximum amount of water leaving the red cells would be 39.9 ml. per 100 ml. of red cells. The data suggest that only about 43 per cent of the red cell water is available for exchange into solutions of increasing tonicity. I N T R O D U C T I O NM a n y studies are available concerning the swdling of erythrocytes in hypotonic solutions (reviewed by Ponder (1-2)). A similar extensive literature on the shrinking of erythrocytes in hypertonic media is not available. The reason for this is twofold: (a) while hematoerit methods are quite accurate in measuring red cell swelling (influx of water), these same methods are inaccurate in measuring red cell shrinkage (efflux of water) and (b) difficulty in accurate correction for trapped interceUular fluid following centrifugation in hypertonic solutions.The purpose of this study is to propose a method of measuring efflux of red cell water based on the change in freezing point depression of the hypertonic media in which the cells are suspended and to determine the quantity of water leaving the red cell in response to measured increases in the tonicity of the surrounding solutions.

Spectrophotometric measurements of the reaction of ferrioxamine B (FeHDFB(+)) with 1,10-phenanthroline (phen) reveal the presence of a ternary intermediate complex in both aqueous solution and an aqueous solution of 0.16 M sodium dodecyl sulfate (SDS). The stoichiometry of the intermediate is Fe(H(2)DFB)(phen)(2+) on the basis of a Schwarzenbach analysis of spectrophotometric data obtained at variable pH and phen concentrations. The ternary complex formation constant for the reaction FeHDFB(+) + H(+) + phen right arrow over left arrow Fe(H(2)DFB)(phen)(2+) is log K = 6.96 in aqueous solution and log K = 8.64 in aqueous 0.16 M SDS. The enhanced stability of Fe(H(2)DFB)(phen)(2+) in micellar solution was analyzed in terms of the pseudophase ion-exchange (PPIE) model of micellar reactions. The association constants for the binding of each reactant to the micellar pseudophase were measured by ultrafiltration. According to PPIE model calculations, the enhanced stability of Fe(H(2)DFB)(phen)(2+) in micellar SDS arises from a proximity effect created by the high local concentrations of reactants in the micellar pseudophase. The calculations also indicate that an inhibitory medium or compartmentalization effect is operative since the observed micellar enhancement is much smaller than predicted by the PPIE model. The micellar stabilization of the Fe(H(2)DFB)(phen)(2+) intermediate and the overall conversion of FeHDFB(+) to Fe(phen)(3)(2+) are discussed as a possible model system for siderophore iron release in microbial organisms.

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