In-situ confocal Raman monitoring of evaporationprocess during protein crystallization

摘要

We have introduced an in-situ Raman monitoring technique to investigate the crystallization process insideprotein drops. In addition to a conventional vapour-diffusion process, a novel procedure which actively stimulates theevaporation from a protein drop during crystallization was also evaluated, with lysozyme as a model protein. In contrastto the conventional vapour-diffusion condition, the evaporation-stimulated growth of crystals was initiated in a simpledehydration scheme and completed within a significantly shorter time. To gain an understanding of crystallization behavioursunder the conditions with and without such evaporation stimulation, confocal Raman spectroscopy combined with linearregression analysis was used to monitor both lysozyme and HEPES buffer concentrations in real time. The confocalmeasurements having a high spatial resolution and good linear response revealed areas of local inhomogeneity in proteinconcentration when the crystallization started. The acquired concentration profiles indicated that (1) the evaporation-stimulated crystallization proceeded with protein concentrations lower than those under conventional vapour diffusion, and(2) crystals under the evaporation-stimulated condition were noticeable within an early stage of crystallization before theprotein concentration approached its maximum value. The HEPES concentration profiles, on the other hand, increasedsteadily towards the end of the process regardless of the conditions used for crystallization. In particular, the observed localinhomogeneities specific to protein distribution suggested an accumulation mechanism of protein molecules that initiatesthe nucleation of crystals.