An improved apparatus has been invented for use in determining the osmotic second virial coefficient of macromolecules in solution. In a typical intended application, the macromolecules would be, more specifically, protein molecules, and the protein solution would be pumped through a flow cell to investigate the physical and chemical conditions that affect crystallization of the protein in question.
Some background information is prerequisite to a meaningful description of the novel aspects of this apparatus. The osmotic second virial coefficient, customarily denoted by the algebraic symbol B22, appears in the equation for the osmotic pressure of a macromolecular solution:
π= RTρ(M – 1 + B22ρ + higher-order terms)
where π is the osmotic pressure, R is the ideal-gas constant, T is the absolute temperature, ρ is the concentration (more specifically, the mass density) of the macromolecule solute, and M is the mass of one mole of the solute. The osmotic second virial coefficient quantifies the degree of attraction or repulsion between the macromolecules under various solution conditions. Therefore, this coefficient is a valuable part of a method of determining optimum conditions for formulation of a protein solution and crystallization of the protein from the solution.
A method of determining B22 from simultaneous measurements of the static transmittance (taken as an indication of concentration) and static scattering of light from the same location in a flowing protein solution was published in 2004. The apparatus used to implement the method at that time included a dual-detector flow cell, which had two drawbacks:
- The amount of protein required for analysis of each solution condition was of the order of a milligram — far too large a quantity for a high-throughput analysis system, for which microgram or even nanogram quantities of protein per analysis are desirable.
- The design of flow cell was such that two light sources were used to probe different regions of the flowing solution. Consequently, the apparatus did not afford simultaneous measurements at the same location in the solution and, hence, did not guarantee an accurate determination of B22. This concludes the background information.
The present improved apparatus includes a flow cell wherein the required simultaneous transmittance and scattering measurements can be made at the same location. For the purpose of these measurements, light from two sources (a laser and an ultraviolet lamp) is delivered simultaneously to the designated location in the cell via a bifurcated optical fiber. The flow cell in this apparatus is narrower than that of the prior apparatus, such that the volume of solution needed for each analysis is of the order of microliters and the mass of protein needed for each analysis at typical concentrations is of the order of micrograms.
The capability of the improved apparatus to yield measurements from which accurate B22 values could be calculated was demonstrated in experiments on several different aqueous lysozyme and concanavalin A solutions for which B22 values had been determined by other means. The apparatus has also been used to screen a series of potential crys albumin, and to evaluate B22 as an indication of the solubility of proteins.