hrvatski

Spinsko obilježavanje ovalbumina

Ovalbumin (OVA) is the major protein in avian egg-white and was one of the first proteins to be isolated in a pure form. It belongs to the serpin (serine protease inhibitors) family but it lacks any protease inhibitory activity. Its ready availability in large quantities has led to its wide- spread use as a standard preparation in studies of the structure and properties of proteins, and in experimental models of allergy [1]. Among the others OVA has been used as a model protein antigen in our investigations of adjuvant effects of immunostimulating peptides [2]. Spin labelling was succesfully applied in the study of interactions of peptidoglycan monomer and structurally related adamantyltripeptides with lipid bilayers in liposomes [3]. The aim of the present study has been to investigate the possible interactions of OVA with immunostimulating peptides and with liposomal bilayers by electron spin resonance (ESR). Ovalbumin was spin labelled with 4-maleimido-2, 2, 6, 6, -tetramethylpiperidine-1-oxyl (TEMPO-maleimide). Spin labelled OVA (SL-OVA) was purified by column chromatography. Fractions containing SL-OVA were concentrated by ultrafiltration on Centricon Centrifugal Filter Devices. The interactions of SL-OVA with phospholipids in liposomal bilayers and with immunological adjuvant peptidoglycan monomer were investigated. The ESR study demonstrated that there was no interaction between OVA as an antigen and peptidoglycan monomer as adjuvant. Also no interactions with liposomal membranes were detected. On the other hand, the reaction of OVA as an antigen and OVA-specific antibodies was recorded as a change in EPR spectra of SL-OVA. Thus it was also demonstrated that spin labelling has no influence on binding of the antibodies to OVA. [1] J.A. Huntington, P.E. Stein, J. Chromatogr. B. 756 (2001) 189-198. [2] J. Tomašić, I. Hanzl-Dujmović, B. Špoljar, B. Vranešić, M. Šantak, A. Jovičić, Vaccine 18 (2000) 1236-1243. [3] R. Frkanec, V. Noethig Laslo, B. Vranešić, K. Mirosavljević and J. Tomašić, Biochim. Biophys. Acta, 1611 (2003) 187-195.

spinsko označavanje; ovalbumin; elektronska spinska rezonancija

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