On-line data acquisition from the ultracentrifuge

The ultraviolet scanner unit of the Beckman model E has been interfaced to a computer for data acquisition. The system is quite simple and the computer data are of comparable accuracy to data obtained from the chart recorder.     

Automated acquisition and analysis of data from the photoelectric scanner of the model E analytical ultracentrifuge.

To facilitate sedimentation equilibrium experiments on the model E analytical ultracentrifuge, a method for automated data acquisition from the photoelectric scanner system has been developed. The instrumentation described is relatively simple to install and convenient to operate. Direct access to computer analysis of data is thus achieved. Application is made to the study of a protein, allophycocyanin. Results over a range of pH and protein concentrations confirm that an α₃β₃ structure is stable in aqueous media.     

Fluorescence detection for the XLI analytical ultracentrifuge

Analytical ultracentrifugation (AUC) provides first-principle hydrodynamic and thermodynamic information concerning the size, shape and interactions of macromolecules. The fundamental measurement needed in AUC is the macromolecular concentration as a function of radial position and time. Currently, the Beckman Coulter XLI analytical ultracentrifuge may be equipped with absorbance and refractive detectors, which provide complementary concentration determinations. For detecting trace quantities of materials, fluorescence detection offers unique advantages over either absorbance or interference detection. A prototype fluorescence detector for the XLI analytical ultracentrifuge has been developed and its characteristics determined. An Ar(+) laser provides a continuous 488-nm excitation beam. Radial resolution is achieved by scanning the focused beam along a radial axis. Detection of the fluorescence signal uses a co-axial, front-face optical configuration to reduce inaccuracies in the concentration caused by inner filter effects. A high-speed A/D data acquisition system allows the fluorescence intensity to be monitored continuously and at a sufficiently high angular resolution so that at any radial position the intensities from all of the samples may be acquired at each revolution. The fluorescence detector is capable of detecting concentrations as low as 300 pM for fluorescein-like labels. The radial resolution of the fluorescence detector is comparable to that of the absorbance system. Both sedimentation velocity and sedimentation equilibrium measurements may be made with the fluorescence detector. Results are presented comparing data acquired using the fluorescence with those acquired using the absorbance detector.     

An optical pulse modulation system for laser interference studies in the analytical ultracentrifuge

The mode of operation and construction of a high-speed laser light modulator is presented that is designed for use with the analytical ultracentrifuge. The system may be used with continuous wave lasers having optical powers not exceeding 100 W and has an optical bandwidth from 200 nm to 800 nm. The electronic modulation circuitry described is capable of producing optical pulses of 0.8 μsec duration at a firing frequency in excess of 100,000 pps and is designed to permit the laser source to operate in either a pulsed or nonpulsed mode. The unit is extremely versatile, and its characteristics permit full advantage to be taken of pulsed laser interferometry in the ultracentrifuge.     

Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques.

Least-squares analysis of experimental data from the analytical ultracentrifuge is discussed in detail, with particular attention to the use of interference optics in studying nonideal self-associating macromolecular systems. Several samples are given that describe the application of the technique, the expected precision of the results, and some of its limitations. A FORTRAN IV computer program is available from the authors.     

DIADEM--a system for the interactive data acquisition and processing in an analytical laboratory.

A conversational program for the acquisition of experimental data in a multi-user, multi-instrument computer system is described. It assists the researcher when recording on-time data. Due to the simple structure of the dialogue, no special knowledge of computer handling is required by the experimenter. Whereas the experimental methods are versatile, a uniform concept of the dialogue and the file structure is realized.     

Histidine-tag-directed chromophores for tracer analyses in the analytical ultracentrifuge

Many recombinant proteins carry an oligohistidine (His(X))-tag that allows their purification by immobilized metal affinity chromatography (IMAC). This tag can be exploited for the site-specific attachment of chromophores and fluorophores, using the same metal ion-nitrilotriacetic acid (NTA) coordination chemistry that forms the basis of popular versions of IMAC. Labeling proteins in this way can allow their detection at wavelengths outside of the absorption envelopes of un-modified proteins and nucleic acids. Here we describe use of this technology in tracer sedimentation experiments that can be performed in a standard analytical ultracentrifuge equipped with absorbance or fluorescence optics. Examples include sedimentation velocity in the presence of low molecular weight chromophoric solutes, sedimentation equilibrium in the presence of high concentrations of background protein and selective labeling to simplify the assignment of species in a complex interacting mixture.     

Challenges for the modern analytical ultracentrifuge analysis of polysaccharides

This article reviews some of the recent advances in analytical ultracentrifugation and how these advances have impacted--and can impact--on our understanding of the size, shape through conformation modelling, interactions and charge properties of polysaccharides in solution, particularly when used in combination with other solution techniques and also imaging techniques. Specifically we look at (1) polysaccharide polydispersity and simple shape analysis by sedimentation velocity, and in particular using new approaches such as SEDFIT analysis; (2) polysaccharide molecular-weight analysis by sedimentation equilibrium and MSTAR analysis and how this complements analysis of size exclusion chromatography coupled to multi-angle laser light scattering; (3) polysaccharide conformation analysis using traditional procedures such as the Wales-van Holde ratio, power law or 'scaling' relations, more specialised treatments for rigid cylindrical structures, semi-flexible chains and worm-like coils and complications through draining effects; (4) Analysis of polysaccharide interactions and in particular complex formation phenomena, focusing on interesting applications in the areas of mucoadhesion and sedimentation fingerprinting; and (5) the possibilities for macromolecular charge and charge screening measurement.     

Analysis of data captured by an on-line image capture system from an analytical ultracentrifuge using schlieren optics

The recent development in this laboratory of an automated data capture system, for refractometric optics on the analytical ultracentrifuge has removed the requirement for tedious and time consuming manual acquisition which had led to a decline in the use of schlieren optics. At the same time this system has increased the amount of data easily available from such an optical system with maintained or increased precision. From the advent of such a system has arisen the need for a package to facilitate the analysis of these data and to extend the range of analytical methods used. Using the improved data sets now available has also enabled us to successfully use methods which have lapsed in popularity over the last two decades. We have also been able to successfully apply radial derivative methods (Bridgman 1942) which have not routinely been applied to the analysis of sedimentation velocity experiments using schlieren optics. In this paper we describe the methods we have so far used to analyse data and present results for previously well defined molecules to demonstrate that the results obtained are reliable. Received: 1 November 1996 / Accepted: 15 January 1997     

Analyses of DNA by automated logging and processing of data from the analytical ultracentrifuge

Methods to log data from buoyant-density and zonal-sedimentation velocity centrifugations of DNA in an analytical instrument are described, as well as computer programs to analyze such data. It is simple and quick to determine modal molecular weights of DNA in neutral or alkaline solutions or buoyant densities in CsCl or Cs₂SO₄. An interactive program which simulates the Dupont curve resolver has also proved useful. Correction equations to convert scanner output to true absorbance were developed.     

A simple test for macromolecular heterogeneity in the analytical ultracentrifuge

A simple check for the presence of heterogeneity in a macromolecular system is proposed, employing comparison of Rayleigh sedimentation-equilibrium patterns for two solutions of the same fringe concentration but differing absolute concentrations. The method is illustrated by application to a bronchial glycoprotein from a cystic-fibrosis patient.     

FUGE: an analytical ultracentrifuge simulation for teaching purposes

An IBM-compatible microcomputer program for teaching purposesis described which simulates the operation of a sedimentationvelocity determination of a protein in an analytical ultracentrifugeusing schlieren optics. The program operates in speeded-up timeand simulates the major procedures which would need to be carriedout to operate such an instrument. The position of the sedimentingboundary can be observed at any time during the run, and upto six ‘photographs’ can be recorded for subsequentanalysis. Calculation of sedimentation coefficient, diffusioncoefficient and mol. wt can be made from a dot-matrix printout.Ten representative proteins are stored within the program, butprovision exists for user-supplied data. Received on June 25, 1987; accepted on September 9, 1987