Obtaining of lysozyme spherulitic forms at ambient temperature using pyrrolidinium octanoat as ionic liquid additive

Abstract

Pyrrolidinium octanoate carboxylate (Py+CnH2n+1COO-; PyO in abbreviation) was used as additive for advanced crystallization of Lysozyme protein, to investigate the impact of protic ionic liquid on the protein crystal morphology. The ionic liquid was synthesized by acidic-base Brönsted neutralization, and its purity was checked by HPLC. The protein crystallization was made through the method of vapour diffusion with hanging drops. Crystallization experiments of Lysozyme with the addition of PyO were performed at 0.4 M PyO and respectively 1.6 M. The morphological of spherulitic forms of Lysozyme in aqueous solutions of PyO protic ionic liquid was investigated by optical microscopy after trials were incubated at ambient temperature (18- 20°C), in various growth periods (3 days and 1 week). Hanging-drop vapour diffusion crystallization experiments with the addition of 0.4 M of PyO show that Lysozyme crystallized in type I spherulitic form. This is assumed to be a result of heterogeneous nucleation, with thin needles radially growing outward from a more or less spherical particle. Hanging-drop vapour diffusion crystallization experiments revealed that the addition of 1.6 M of PyO led to a second type of spherulitic form of the Lysozyme.