A spectrophotometer-based method for crystallization induction time period measurement

A method for measurement of crystallization induction time periods using a spectrophotometer is described. The turbidity of lysozyme solutions at sodium chloride concentrations of 0%, 3%, 6%, 9% (w/v) in 0.1 M NaAc (pH 4.0) and a lysozyme concentration of 10 mg/ml was monitored at 350 nm and showed a dramatic increase after nucleation occurred. Heterogeneous sources of nucleation were removed via filtration with 100,000 and 500,000 MWCO filters, Larger pore size, 0.2 mum, filters were not able to remove these particles. The non-filtered and 0.2 mum filtered had the same turbidity profiles except that the Ultra filtered had an induction time of 152 min compared with the 119 min induction time for the non-filtered solution. The 500,000 MWCO filter had a significant reduction in the formation of prenucleation aggregates and the induction time increased from 119 min for non-filtered to 178 min for the 500,000 MWCO filtered. Also, when the 100,000 MWCO filter was used there was no nucleation or crystallization over the 240 min time frame. The increase in turbidity was attributed to an increase in particle size and not to bacterial growth because the solutions were found to be free of bacteria or fungi by microbiological analysis. Results from hanging drop vapor diffusion crystallization on the same solutions used in the turbidity study indicate that the sources removed by filtration lead to nucleation. Finally, sodium azide was added to the solutions at a concentrations of 0.17% (w/w) and was found to interfere with or delay lysozyme nucleation. (C) 2001 Elsevier Science B.V. All rights reserved.