UV laser scanning and fluorescence monitoring of analytical ultracentrifugation with an on-line computer system.
A new optical system for the analytical ultracentrifuge is described, which permits sedimentation to be monitored by fluorescence. The optical system is based on a laser light source, which is focused to a narrow (50 micron) beam. The radical scanning of the beam provides information on the distribution of fluorescing material with distance in the centrifuge cell. Data collection and processing are performed in conjunction with an on-line computer system which sorts incoming fluorescence pulses according to rotor hole and cell sector, averages families of pulses to improve signal to noise ratios and fits the data (in the experiments reported here) to equations to determine sedimentation coefficients. Initial experiments with the system have been performed with bovine serum albumin and indicate that sedimentation can be readily monitored by fluorescence with solutions at concentrations as low as 20 micrograms per ml, with excitation at 257 nm. At these concentrations, the optical density is only in the 0.01 range, too low for experiments with absorption-scanner optical system. Even lower concentrations can be used when fluorescent labels are used with excitation in the visible region of the spectrum. The preliminary studies indicate that fluorescence monitoring of sedimentation will substantially enhance the range of experimental possibilities in ultracentrifugation by improving both the sensitivity of measurements and the discrimination between sedimenting species on the basis of their fluorescence characteristics.
D003201:Computers; D007834:Lasers; D009862:Online Systems; D011487:Protein Conformation; D012710:Serum Albumin, Bovine; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation / Q000379:methods*; D014461:Ultracentrifugation / Q000295:instrumentation / Q000379:methods*
D012710:Serum Albumin, Bovine