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1.
Chemphyschem ; 10(9-10): 1492-9, 2009 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-19360797

RESUMO

We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm(-1) to 1632 cm(-1), indicating a shift from alpha-helical structure to beta-sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to beta-sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.


Assuntos
Enzimas Imobilizadas/química , Subtilisinas/química , Dicroísmo Circular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Triptofano/química
2.
Chem Commun (Camb) ; (17): 1880-1, 2002 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-12271655

RESUMO

The temperature dependence of the bimolecular rate constants for a diffusion controlled reaction involving neutral reactants have been directly determined in five commonly used ionic liquids over the temperature range 5-70 degrees C.

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