Photoaffinity labelling with fluorescence detection

Abstract

A micromethod was developed for investigating the interactions between fluorescent dyes and cellular proteins. The lipophilic cationic dye APMC (azopentylmethylcarbocyanine) contains a photosensitive diazirine ring and is suitable for photoaffinity labelling. By combining photoaffinity labelling of cultured cells, micro-gel electrophoresis and detection of the fluorescence with a microfluorimeter, we established a highly sensitive and rapid procedure to identify APMC labelled proteins. Cells which had been incubated for 10 min with 10(-8) M APMC could be analysed for APMC binding without difficulty. Under our experimental conditions this corresponds to about 0.2 nmol APMC per mg protein. The lipophilic APMC specifically stains the mitochondria in living HeLa and LM cells. The fluorescing mitochondria can be easily detected under a fluorescence microscope. By photoaffinity labelling we were able to show that at low dye concentrations APMC preferentially marks four proteins with apparent molecular masses of 31, 40, 66, and 74 kDa. In order to establish that these are mitochondrial proteins, we isolated and analysed the mitochondria from incubated HeLa and LM cells; again, the same four proteins were detected. They are most probably proteins of the inner mitochondrial membranes, which accumulate the lipophilic APMC cations.