Liquid diffusion studies: tracer and optical methods

Abstract

A capillary cell diffusiometer was designed, developed and used to measure diffusion coefficients of highly complex systems that are of biological interest. Tracer diffusion coefficients of aqueous urea solutions at 25°C in the concentration range 0.3 M to 3.1 M were measured with the diffusiometer. These diffusion coefficients, having been previously accurately measured by another method, were used to calibrate the capillary cell diffusiometer. After calibration of the diffusiometer, tracer diffusion coefficients of 1.0 M urea in buffered urea - alpha-chymotrypsin solutions at 25°C were measured. The diffusion results were used to elaborate on a possible process by which urea causes protein denaturation. Also the diffusion measurements were used to discuss the possibility that urea prohibits alpha-chymotrypsin dimerization. Under solution conditions where enzyme dimers should have formed, none were found by viscosity measurements. The urea diffusion measurements indicated that instead of enzyme dimers forming, the urea molecules were associating with the alpha-chymotrypsin molecules. Using the capillary cell diffusiometer, tracer diffusion coefficients of three bile acid solutions -- cholic, taurocholic and taurodeoxycholic -- and four fatty acid solutions -- acetic, pentanoic, octanoic and decanoic -- were measured. The diffusion systems contained the bile acids or fatty acids over the concentration range of 0.12 mM to 1.0 mM. Each experimental solution contained 0.01 M sodium phosphate (pH 7.1) and 0.14 mM NaCl. All measurements -- tracer diffusion coefficients, densities and viscosities -- were determined at 37°C. The same measurements at 37°C were also performed on the aqueous system, sucrose - 0.9% NaCl, over the sucrose concentration range from 0.05 M to 0.53 M. As a continuation of diffusion studies of systems of biological interest, the ternary diffusion coefficients at 37°C of a series of amino acid-NaCl-H20 systems were measured. Two mean solute concentration regions were studied, 0.5 M and 1.0 M. The series of amino acids were glycine, alanine and alpha-aminobutyric acid. These diffusion experiments were performed by the optical Rayleigh interference method. A computer-controlled scanning microcomparator for the analysis of the experimental interference fringes recorded on photographic plates was designed, manufactured and used for these ternary systems. No previous theoretical treatment was available for the determination of ternary diffusion coefficients by the Rayleigh interference method, therefore a theoretical treatment was developed and used for their determination.