| Popis: |
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. |
