RATE ZONAL SEDIMENTATION OF PROTEINS IN ONE-HOUR OR LESS

Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein-protein interaction s. However, rate zonal sedimentation experiments typically last approx imately 1 day. In contrast, this report describes a rate zonal sedimen tation method requiring 1 h or less. This was accomplished by centrifu ging small density gradients (200 mu l) prepared with sucrose or OptiP rep in a fixed-angle rotor at high relative centrifugal force, By usin g small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. F or a variety of proteins, plots of S-20,S-w versus distance sedimented during centrifugation in a TLA 120.2 rotor were linear, As a practica l application, sedimentation of the heterotrimeric stimulatory G prote in and its dissociated alpha-subunit were determined. The results were similar to those obtained with 17- to 22-h centrifugations in an SW 5 0.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein-protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out t raditional rate zonal sedimentation experiments, A rate zonal sediment ation technique that rivals many chromatographic methods in celerity w ill help to circumvent these problems. (C) 1997 Academic Press.