The determination of diffusion constants of proteins by a refractometric method

Abstract

IN the calculation of the molecular weights of proteins from sedimentation data it is essential that their diffusion constants should be known accurately. At first diffusion constants were measured from the spreading at the sedimenting boundary in the ultracentrifuge [Svedberg, 1925, 1, 2] and the concentration changes were determined from measurements of the light absorption of the solutions. But in the ultracentrifuge it is impossible to control the temperature to a degree necessary for accurate diffusion measurements. A further objection to this method of measuring diffusion constants is that the time of diffusion in the centrifuge cell is far too short for an extended series of measurements. Therefore Tiselius and Gross [1934] applied the light absorption method to the measurement of duffusion at a boundary in a tube at rest. Small fluctuations in the results persisted due to variation of the photographic blackening caused by irregularities in the photographic emulsion or by traces of impurities in the solutions. In the work to be described in this paper the diffusion apparatus was similar to that used by Tiselius and Gross but the concentration gradients were measured by a refractometric method developed by one of us for use in the ultracentrifuge [Lamm, 1928; 1929]. In this method a uniform transparent scale is photographed through the diffusion cell. The refractive index gradient at the diffusion boundary produces a distorted image of the scale in which scale line displacement is proportional to the concentration gradient when the refractive index is a linear function of the concentration. The arrangement of the apparatus is shown in Fig. 1. Fig. 1. Diffusion apparatus. C represents the scale and D the diffusion tube. A is a quartz mercury arc lamp, B a filter which transmits light of a wavelength not absorbed by the solution examined, C the scale, D the diffusion cell in a water thermostat E, and F the camera. The camera objective has a focal length of 100 cm. and is placed in such a position that the scale image has a magnification of about one. (528)