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Analytical ultracentrifugation (AUC) provides the most widely applicable, precise, and accurate means for characterizing solution hydrodynamic and thermodynamic properties. While generally useful, boundary sedimentation velocity AUC (SV-AUC) analysis has become particularly important in assessing protein aggregation, fragmentation and conformational variants in the same solvents used during drug development and production. In early 2017 the only manufacturer of the analytical ultracentrifuge released its newest analytical ultracentrifuge, the Optima, to replace the aging second-generation XLA/I series ultracentrifuges. However, SV-AUC data from four Optima units used in the characterization of adeno-associated virus (AAV) have shown evidence of sample convection. Further investigation reveals this problem arises from the design of the temperature control system, which makes it prone to producing destabilizing temperature-induced density gradients that can lead to density inversions. The problem is intermittent and variable in severity within a given Optima unit and between Optima units. This convection appears to be associated mainly with low rotor speeds and dilute concentration of solvent components, i.e., AAV analysis conditions. Data features diagnostic for this problem and strategies for its elimination or minimization are provided.

Several studies have shown that divalent anion binding to ribonuclease A (RNase A) contributes to RNase A folding and stability. However, there are conflicting reports about whether chloride binds to or stabilizes RNase A. Two broad-zone experimental approaches, membrane-confined electrophoresis and analytical ultracentrifugation, were used to examine the electrostatic and electrohydrodynamic characteristics of aqueous solutions of bovine RNase A in the presence of 100 mM KCl and 10 mM Bis-Tris propane over a pH range of 6.00-8.00. The results of data analysis using a Debye-Huckel-Henry model, compared with expectations based on pK(A) values, are consistent with the binding of two chlorides by RNase A. The decreased protein valence resulting from anion binding contributes 2-3 kJ/mol to protein stabilization. This work demonstrates the utility of first-principle valence determinations to detect protein solution properties that might otherwise remain undetected.

Analytical ultracentrifugation is a widely used method for characterizing the solution behavior of macromolecules. However, the two commonly used detectors, absorbance and interference, impose some fundamental restrictions on the concentrations and complexity of the solutions that can be analyzed. The recent addition of a fluorescence detector for the XL-I analytical ultracentrifuge (AU-FDS) enables two different types of sedimentation experiments. First, the AU-FDS can detect picomolar concentrations of labeled solutes, allowing the characterization of very dilute solutions of macromolecules, applications we call normal use tracer sedimentation (NUTS). The great sensitivity of NUTS analysis allows the characterization of small quantities of materials and high-affinity interactions. Second, the AU-FDS allows characterization of trace quantities of labeled molecules in solutions containing high concentrations and complex mixtures of unlabeled molecules, applications we call biological on-line tracer sedimentation (BOLTS). The discrimination of BOLTS enables the size distribution of a labeled macromolecule to be determined in biological milieus such as cell lysates and serum. Examples that embody features of both NUTS and BOLTS applications are presented along with our observations on these applications.

The approaches presented in this series of papers make possible rapid gathering, reduction, and analysis of data from the Rayleigh interference optical system of an analytical ultracentrifuge. Instrumentation described in this paper provides some of the timing and measurement circuits necessary for a microprocessor or minicomputer to determine the rotor frequency, rotor period, and elapsed time of an experiment. It includes simple but effective circuits to generate precise rotor timing pulses that are useful for synchronization of pulsed light sources. Circuits to control photographic operations in the ultracentrifuge are described briefly. All of these circuits are interfaced to a simple microcomputer address/data bus. An adapter between this bus and a Q-bus (for a DEC LSI - 11 2 or LSI 11 23 microcomputer) is also described. The circuits presented have been used in this laboratory over a 3-year period. They have proven reliable and form an integral part of the real-time data acquisition systems that have been constructed.

Charge is a fundamental property of macromolecules in solution. However, estimation of the apparent charge on polyions has confounded science for decades. Presented here is a general method to determine directly the apparent charge on a polyion, regardless of its size or shape. This new method uses equilibrium electrophoresis, a procedure in which opposing solute flows from electrophoresis and from diffusion balance everywhere as the system reaches a steady-state distribution. The method uses only small quantities of materials, is nondestructive, and requires only simple, inexpensive instrumentation. Here we describe a prototype apparatus, demonstrate the phenomenon, and present experimental examples of the procedure.