Determination of the sedimentation coefficient and molecular weight of proteins by density gradient ultracentrifugation in fixed angle rotor.

Abstract

The applicability of density gradient ultracentrifugation using fixed angle rotors for the determination of sedimentation coefficient and molecular weight of proteins was studied. Ovalbumin, bovine serum albumin, IgG-, IgA- and IgM-globulin proteins, as standards were, among others, centrifuged in a fixed angle rotor (Beckman type 50) in 5-20% (w/v) sucrose density gradient for 300 min, at a speed of 40,000 rpm, at 5 degrees C. After centrifugation, the sedimentation diagram of the above-mentioned proteins was taken by a spectrophotometer equipped by a flow-through cuvette and a compensator. The sedimentation path was measured on the diagrams. A calibration curve was drawn by plotting the sedimentation coefficients of the standard proteins against the sedimentation paths. This curve, for which the equation y = 1.74x-1.6 is valid, was used for the determination of the sedimentation coefficients of several globular proteins under the conditions described above. The values obtained this way agree within +3-10% with those determined with analytical ultracentrifuge. If the logarithms of proteins molecular weight used as standards were drawn against the logarithms of their sedimentation paths a calibration curve was obtained. This curve for which the equation ln y = 2.022 ln x + 8.71 is valid can be used for the determination of molecular weights of globular or nearly globular proteins.