A simple numerical method for the construction of isokinetic sucrose density gradients, and their application to the characterisation of immunoglobulin complexes

Abstract

A simple numerical method for calculating the parameters required for the production of isokinetic sucrose density gradients is described. Calculation is based on tables of viscosity and density of sucrose solutions at various concentrations. When density gradients prepared according to this method were used for centrifugation of a number of well characterised proteins, it was shown that isokinetic behaviour occurs, and that sedimentation coefficients obtained are comparable to those determined by conventional analytical ultracentrifugation. The method was used to characterise polymers of IgM and IgG, and results suggest that IgM polymers up to the tetramer can occur in Waldenstrom's macroglobulinaemic serum, and that IgG polymers up to the pentamer can occur in stored specimens of ethanol precipitated human IgG. The sedimentation behaviour of 125I-IgE was studied by fractionating the gradients and counting the fractions, and it was shown that degradation of the labelled IgE took place during storage. It was also found that labelled IgE induces rapid breakdown of IgG with which it was mixed prior to centrifugation. The method has also been used to study immunoglobulin-containing complexes found in the knee joint fluids of patients with rheumatoid arthritis. The high degree of resolution of this method is demonstrated by the fact that many more of these components were detectable by density gradient centrifugation than by conventional analytical ultracentrifugation.