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Sensitivity of obelin and apoobelin to digestion by some proteases Ludmila A. Frank; Vladimir S. Bondar; Eugene S. Vysotski* Institute of Biofisics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia      *(vys@bmbiris.bmb.uga.edu) In a cooperation with Bayer AG, recombinant obelin was developed as a probe for monitoring calcium free concentration in mammalian cells. To interpret correctly the light signal obtained from cells we have determined the influence of some environmental conditions on the luminescent properties of photoprotein. To estimate possible consequences of digestion by proteases on the luminescent activity, proteases of different substrate specificity (trypsin, papain, Staphylococcus aureus strain V8 protease, aminopeptidase M, and carboxypeptidase Y) were tested. Apo- and photoprotein digestion was carried out in a buffer containing 150 mM KCl, 5 mM PIPES pH 7.1 (and 2 mM EDTA in the case of photoprotein) at 37 and 4oC. Photoprotein bound with coelenterazine is more stable to digestion with any tested proteases than apophotoprotein. The residual luminescent activity of obelin after 16 h of incubation with proteases lies in the range from 100% (on digestion by carboxypeptidase Y) to 60% (on digestion by S.aureus strain V8 protease). Most dramatically the luminescent activity drops during the first 2 h of incubation. For comparison the same experiments were carried out with aequorin and apoaequorin. Supported by Bayer AG (Germany). [Poster: vysotski.eugene.25132]

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