Centrifugation optical systems

Selected entries from Methods in Enzymology [vol, page(s)] Association constant determination, 259, 444-445 buoyant mass determination, 259, 432-433, 438, 441, 443, 444 cell handling, 259, 436-437 centerpiece selection, 259, 433-434, 436 centrifuge operation, 259, 437-438 concentration distribution, 259, 431 equilibration time, estimation, 259, 438-439 molecular weight calculation, 259, 431-432, 444 nonlinear least-squares analysis of primary data, 259, 449-451 oligomerization state of proteins [determination, 259, 439-441, 443 heterogeneous association, 259, 447-448 reversibility of association, 259, 445-447] optical systems, 259, 434-435 protein denaturants, 259, 439-440 retroviral protease, analysis, 241, 123-124 sample preparation, 259, 435-436 second virial coefficient [determination, 259, 443, 448-449 nonideality contribution, 259, 448-449] sensitivity, 259, 427 stoichiometry of reaction, determination, 259, 444-445 terms and symbols, 259, 429-431 thermodynamic parameter determination, 259, 427, 443-444, 449-451. 

The sedimentation equilibrium experiment requires much smaller volumes of solution, about 0.15 ml. With six-hole rotors and multichannel centerpieces (41) it is potentially possible to do fifteen experiments at the same time. For situations where the photoelectric scanner can be used one might (depending on the extinct coefficients) be able to go to much lower concentrations. Dust is no problem since the centrifugal field causes it to go to the cell bottom. For conventional sedimentation equilibrium experiments, the analysis of mixed associations under nonideal conditions may be virtually impossible. Also, sedimentation equilibrium experiments take time, although methods are available to reduce this somewhat (42, 43). For certain situations the combination of optical systems available to the ultracentrifuge may allow for the most precise analysis of a mixed association. The Archibald experiment may suffer some loss in precision since one must extrapolate the data to the cell extremes (rm and r6) to obtain MW(M, which must then be extrapolated to zero time. Nevertheless, all three methods indicate that it is quite possible to study mixed associations. We have indicated some approaches that could be used to overcome problems of nonideality, unequal refractive index increments, and unequal partial specific volumes. 

An ultracentrifuge is a high-speed centrifuge equipped with a suitable optical system (usually schlieren or interference optics, the latter being particularly useful when low concentrations are involved) for recording sedimentation behaviour and with facilities for eliminating the disturbing effects of convection currents and vibration. The sample is contained in a sector-shaped cell mounted in a rotor (usually c. 

The instrument routinely used in our lab for AUC experiments is the Beckman XL-I analytical ultracentrifuge. We exploit the UV absorption property of RNA molecules at 260 nm to employ the absorbance optics built in the centrifuge. The optical system of a Beckman analytical ultracentrifuge is shown schematically in Fig. 10.1A. The instrument is... 

Figure 1 Sedimentation velocity analysis of the interaction between bisANS and bacteriophage P22 coat protein. Sedimentation was carried out at 56,0(X) rpm at 20 °C in a Beckman Model E centrifuge equipped with a video-based on-line Rayleigh optical system. (TOP) Coat protein alone (cq = 0.4 mg/ml t = 5348 sec) (BOTTOM) Coat protein in the presence of 60 (iM bisANS (A) Co = 0.5 mg/ml t = 5339 sec (B) 0.2 mg/ml t = 5343 sec). The error bars are the standard error of the mean propagated from the averaging process.

Liu, S. and W. F. Stafford (1992). A Real-Time Video-Based Rayleigh Optical System for an Analytical Ultracentrifuge Allowing Imaging of the Entire Centrifuge Cell. Biophys. J. 61, A476, 2745. 

Figure 2 9 Sedimentation velocity measurement, (a) The distribution of protein in the cell a function of centrifugation time. The protein is sedimenting toward the right. (6) Schlien optics pattern. The optical system measures the change in refractive index of the soluiic Thus, the pattern gives the concentration gradient of protein along the sedimentation path, i Plot of log X versus time where x is the distance the boundary has moved (i.e., the distance frc the meniscus to the peak of the SchSeren pattern).

Sedimentation equilibrium methods measure the concentration gradients of molecules in solution when spun at high speed in a centrifuge. Samples are held in special cells in the rotor, with optically flat clear windows and, in current instruments, several samples (including controls) may be run simultaneously. The concentration gradient, as a function of radius (r), is measured using UV/visible absorbance, fluorescence or refractive index methods, using optical systems mounted outside the rotor. 

AUC instrumentation consists of a centrifugal drive unit, a rotor in which the sample is held, one or more optical systems to observe the sample, and a computer to acquire and to analyze the data. 

Fig.3. Sedimentation pattern of normal human serum in the ultracentrifuge, measured with a schlieren optical system. Left hand picture taken 51 min after start of centrifugation right hand picture after 125 min. Centrifugation at 59,800 rpm. A albumin, 4.5S G = globulin, 7S M = macroglobulin, 19S.

There are several commercial photosedimentation analyzers. Figure 34-8 shows a schematic diagram of one instrument (the Horiba CAPA-700) that can achieve sedimentation by gravity or by centrifugal force. The components consist of an optical system, a centrifuge, control circuitry, and a computation system. 

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