Effect of large refractive index gradients on the performance of absorption optics in the Beckman XL-A/I analytical ultracentrifuge: an experimental study Jos  e Manuel Gonz alez, a Germ an Rivas, a and Allen P. Minton b, * a Centro de Investigaciones Biol ogicas, Consejo Superior de Investigaciones Cient  ıficas, 28006 Madrid, Spain b National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Received 19 August 2002 Abstract An analytical centrifuge cell was modified to detect refraction of light transmitted through the cell caused by refractive index gradients formed by sedimenting solute during centrifugation. Sedimentation velocity and sedimentation equilibrium experiments were carried out in this cell on solutions containing high concentrations of protein and polysaccharide in a Beckman–Coulter XLA analytical ultracentrifuge. Analysis of the results indicates that in the absence of an optical artifact easily recognized as a ‘‘black band,’’ the dependence of apparent absorbance upon radial position reported by the instrument may be considered a reliable measure of the solute concentration gradient. Ó 2003 Elsevier Science (USA). All rights reserved. Workers in the field of analytical ultracentrifugation have long been familiar with the phenomenon called ‘‘Wiener skewing,’’ in which light entering a centrifuge cell is refracted away from the path of entry by the presence of a gradient of refractive index that develops as solutes sediment within the centrifuge cell [1–3]. When the radial gradient of refractive index becomes sufficiently large, the light exiting the sample cell devi- ates so greatly from the path of incoming light that it does not enter the detector slit facing the light source, which the detector reports as a radial segment of infinite absorbance or a ‘‘black band.’’ This situation is depicted schematically in Fig. 1. Because the characterization of macromolecular interactions requires the study of macromolecular solutions over broad ranges of total solute concentration [4,5], we were concerned that at the highest solute concentrations employed in such studies, refractive index gradients formed within the sample might result in deviations between the true and the ap- parent (reported) absorbance gradients in the absence of an obvious black band, which could introduce errors into the interpretation of the apparent gradients. The detailed analysis of this phenomenon is quite complex [1–3] and depends critically on the precise geometry and properties of the optical instrumentation employed in the analytical ultracentrifuge and on the magnitude of the refractive index gradient. Hence the extent to which angular deviation of light resulting from Wiener skewing might distort a measured gradient in the absence of an obvious black band is not readily predictable on theo- retical grounds. We therefore adopted an empirical ap- proach to the study of this effect and its possible influence on absorbance gradients obtained from sedi- menting solutions of moderately concentrated macro- solutes. Materials and methods Materials Ovalbumin, purchased from Sigma (St. Louis, MO) was dissolved in 50 mM sodium phosphate, 100 mM NaCl buffer, pH 7.5, and dialyzed extensively against buffer. Dextran (40,000 kDa), obtained from Sigma, was dissolved in water. Analytical Biochemistry 313 (2003) 133–136 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: 1-301-402-0240. E-mail address: minton@helix.nih.gov (A.P. Minton). 0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. PII:S0003-2697(02)00434-7