A method for the measurement of the sedimentation coefficient and molecular weight of microgram quantities of proteins in 6 m guanidine hydrochloride

A technique has been perfected for measuring the sedimentation coefficient of microgram quantities of a reduced protein in 6 m guanidine hydrochloride. The protein is sedimented through a gradient of 5-8 m guanidine-HCl in the presence of dithiothreitol in a SW 50.1 swinging-bucket rotor. Run conditions are calibrated by a simultaneous measurement using a single reference protein. Thus, the need for running a calibration curve involving several standard proteins simultaneously with a sample is eliminated. Because of the trace quantity of protein used, the technique yields an estimate of the sedimentation coefficient at zero concentration (s⁰) directly without extrapolation. Since s⁰ is a function of the molecular weight of a reduced protein in this solvent, the method also allows an estimate of the subunit molecular weight of the protein. The results of the application of the method to known proteins are reported.