The aim of the study was to determine the type and activity of body-surface proteases of live and dead workers. Samples were collected for five weeks. 100 samples of live and dead worker bees were gathered, respectively, each containing 10 bees. The total number of samples was 200. Hydrophilic (water-treated) and hydrophobic (Triton-rinsed) proteins were isolated from the insects. The rinsing samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by a modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; and electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor detection by means of a modified Felicioli method; and in vivo tests of antifungal and antibacterial activity using the double application method. The concentration of hydrophobic proteins on the body surface of the bees was found to be higher than that of hydrophilic proteins. The activity of proteases and their inhibitors remained at a steady level in the dead bees during the five weeks, whereas that of the live bees was variable. The dead workers were found to have aspartic, serine, thiolic and metallic proteases on the body surface; the live bees, in turn, had aspartic and serine proteases. The dead bees were less resistant to microorganisms. The methods used in the present study can be employed for assessing the condition and state of health of bee colonies, both prior to and after wintering, as well as during the beekeeping season.
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