The Astbury Centre for Structural Molecular Biology

Managers:

Dr Iain Manfield
i.manfield@leeds.ac.uk
0113 343 7279

Amy Magoolagan
a.magoolagan@leeds.ac.uk
0113 343 7278

Introduction

The instruments can be used to determine the following parameters in free solution:

• distribution of species (s or M): heterogeneity
• degree of aggregation; whether reversible
• the stoichiometry and affinities of interacting macrosolutes
• MW in solution of distinct species; diffusion coefficients
• overall hydrodynamic shape of distinct species

The AUC is used in two modes:

1. Sedimentation velocity
2. Sedimentation equilibrium

Sedimentation velocity is performed at high rotor speed (typically 30 - 60000rpm) for 1 to 6 hours. It determines the rate of movement of macrosolutes within a centrifugal field and provides a rapid means for measuring several hydrodynamic parameters. It allows calculation of sedimentation coefficient(s), diffusion coefficients and molecular weights, provided that the sedimenting components are well separated, shape information (estimation of particle asymmetry). Interacting systems can be characterised if several concentrations are spun in the same rotor. Velocity Data

Sedimentation equilibrium is performed at lower rotor speed such that solute particles do not pellet at the bottom of the cell but redistribute over time, with an increasing concentration toward the outer radius of the cell. After a certain length of time (several hours to several days) the effect of centrifugal force on the solute particles is balanced by diffusion at every point in the cell, and the apparent concentration does not change. This technique allows calculation of molecular weight, homogeneity with respect to MW, association constants and aggregation states. Data from velocity experiments can be combined with equilibrium data to perform global analysis.

XL-I Specifications

Wavelength range: 190 - 800nm

radial scans at two chosen wavelengths possible
simultaneous interference scan is usually performed.

MW range: 10² - 5 x 10⁶ Da.

Concentration range(Absorbance): 0.05mg/ml - 2mg/ml (80mg/ml or more using shoulder of peak)

(Interference): 0.025mg/ml - 30mg/ml

Binding (Dissociation) constant range: K_D of 10^-3 - 10^-8 M

There are two XLI analytical ultracentrifuges in the Wellcome Trust Centre for Biomolecular Interactions located on level 9 of the Astbury Building. Both instruments are equipped with absorbance and interference optics. We have 4-place and 8-place rotors, and 2-sector and 6-sector cells with a choice of quartz or sapphire windows.

Optical systems

The absorbance system is essentially a double-beam spectrophotometer set into the base of the centrifuge chamber coupled to a slit which scans the cell from inner to outer radius. Its advantages are selectivity (e.g. ability to discriminate between protein and nucleic acid), true baseline determination, and tolerance of imperfectly matched reference buffer. The disadvantages of absorbance scanning are the long time interval (90 sec to scan one cell) and the relatively noisy signal.

The Rayleigh interference system detects solute concentration differences by changes in refractive index. The advantages are speed (10sec to scan one cell) and excellent signal-to-noise ratio. Interference and absorbance scans can be performed at the same time enhancing selectivity, but losing the speed advantage of the interference system. Because even small molecules form steep refractive index gradients during centrifugation it is essential that sample and buffer have equal chemical potential, usually achieved by dialysis. For the same reason the column heights in sample and reference sectors of the centrifuge cells need to be equal. Another disadvantage is that in order to determine the baseline (needed for equilibrium experiments) a separate run has to be performed with the same cells rinsed out and refilled with water without disassembling the cells.

Information on sample preparation is found here

Booking instrument time, and submission of samples

A booking diary is maintained by Amy Magoolagan. To arrange time slots contact her, either contact by email or personally in Room 9.108g, Astbury Building

The facility is run as a service. You provide the samples and reference buffer; we perform the experiment and analyse the results. However those wishing to get more involved in the experiments are welcome to set up and operate the centrifuge following training. User-analysis of the data generated, and experimental design are also encouraged. Up to date software can be downloaded from the RASMB web site. http://www.bbri.org/RASMB/rasmb.html

Before a run is set up, a number of details are required to enable appropriate conditions to be used in the experiment, and to ensure proper labelling of the resulting data. A sample submission form is available electronically. A modest access charge is levied to help pay for the annual maintenance costs.

We ask that the filled-in form is returned electronically at least 24 hours before the experiment so that equipment can be prepared. Results are returned as email attachments in the form of a Word document or an Excel spreadsheet. The filled-in form can be embedded in these files to save needless re-typing of details, and to ensure the results can be understood when retrieved from the archives. The raw data can occupy as much as 100 MB. These large files can be copied to CD or transferred via the local network.

All publications arising from use of this facility should acknowledge the funding provided by the Wellcome Trust under the Joint Infrastructure Fund (JIF) initiative.