Optical spectroscopic methods for probing the conformational stability of immobilised enzymes

<b>Structure of immobilised enzymes</b>: Circular dichroism, infrared and fluorescence spectroscopic methods are used to characterise in situ structural stability of enzymes immobilised on particles. The enzyme <i>subtilisin Carlsberg</i> exhibits a similar secondary structure in solution and in the immobilised state but on the surface of porous silica gel a rigid tertiary structure is preferred. 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<sup>-1</sup> to 1632 cm<sup>-1</sup>, indicating a shift from α-helical structure to β-sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to β-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

Profissionais da área da saúde: conhecimento e terminologia de documentação Professionals of the health area: knowledge of the terminology of documentation

A interação dos profissionais das áreas da saúde e de informação permitirá não só consolidar o processo de comunicação, mas também agilizar as várias etapas da geração de novos conhecimentos. Para isso, é necessário dominar a terminologia considerada básica para compreender o fluxo de comunicação e os termos essenciais ao acesso à informação, quer pelo processo manual e/ou automatizado. Os resultados alcançados em uma pesquisa realizada com uma comunidade da área de saúde permitiram a adoção de estratégias que iriam facilitar a preparação desses pesquisadores para participar das etapas posteriores de pesquisa mais abrangente, que está sendo efetuada para implantação de um "Centro de Informação e Documentação". Foram comparadas as respostas da comunidade da área da saúde com as de uma elite selecionada de uma comunidade da área tecnológica.<br>The interaction between the professionals of the health and information fields will not only open up the possibility of consolidating the communication process, but also will lead to the spreding up of the various stages of the generation of further knowledge. With a view, to understanding the communication flux and the essential terms for access to information (either manual and/or automatized) it is necessary to dominate the basic terminology. The results achieved by a research project undertaken with researchers of the health field led to the adoption of strategies which should facilitate the preparation of these researchers for their participation in later stages of more comprehensive research with a view to the establishment of an Information and Documentation Center. The answers of this group were compared to those of the select elite of a community of the technological field