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Volume 34 Number 3 Volume 34 Number 4 Volume 35 Number 1

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Research articles

ScienceAsia 34 (2008): 400-408 |doi: 10.2306/scienceasia1513-1874.2008.34.400


In-situ confocal Raman monitoring of evaporation process during protein crystallization


Jitraporn˙Vongsvivuta,b,*, Shu˙Watanabea, Kohki˙Nodaa, Hidetoshi˙Satoa, Hideo˙Tashiroa

 
ABSTRACT:     We have introduced an in-situ Raman monitoring technique to investigate the crystallization process inside protein drops. In addition to a conventional vapour-diffusion process, a novel procedure which actively stimulates the evaporation from a protein drop during crystallization was also evaluated, with lysozyme as a model protein. In contrast to the conventional vapour-diffusion condition, the evaporation-stimulated growth of crystals was initiated in a simple dehydration scheme and completed within a significantly shorter time. To gain an understanding of crystallization behaviours under the conditions with and without such evaporation stimulation, confocal Raman spectroscopy combined with linear regression analysis was used to monitor both lysozyme and HEPES buffer concentrations in real time. The confocal measurements having a high spatial resolution and good linear response revealed areas of local inhomogeneity in protein concentration 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 the protein concentration approached its maximum value. The HEPES concentration profiles, on the other hand, increased steadily towards the end of the process regardless of the conditions used for crystallization. In particular, the observed local inhomogeneities specific to protein distribution suggested an accumulation mechanism of protein molecules that initiates the nucleation of crystals.

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a RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
b Centre for Biospectroscopy, School of Chemistry, Monash University, Wellington Road, Clayton, Victoria 3800, Australia

* Corresponding author, E-mail: jitraporn.vongsvivut@sci.monash.edu.au

Received 3 May 2008, Accepted 3 Nov 2008