Diffusive interfacial transport: A new approach to concentrated protein solution studies

A spatial gradient in the concentration of binary protein-water systems is deliberately created by slow evaporation of a solution of solute. Such samp les are scanned by optical interferometry to record the refractive index pr ofile and by near-infrared microspectroscopy to determine the water composi tion along the spatial gradient. Combination of the two data sets allows th e change in refractive index with concentration and its increment dn/dc to be determined beyond the usual report limited to low protein-solvent volume fractions. Further, phase behavior of protein solutions can be studied. To illustrate the method, cytochrome c is chosen; clean, bubble-free samples of extremely concentrated (ca. 80%) metastable solutions can be produced in this way, and in time, protein crystals separate from these solutions.