The Isolation and Properties of a Lipoprotein Fraction Possessing Tyrosinase Activity from the Haemolymph of the Larva of the fleshfly, Sarcophaga barbarta

Haemolymph from late third-instar larvae of the blowflies Calliphora sp. (Karlson & Schweiger, 1961 ; Thomson & Sin, 1970) and Lucilia cuprina (Hackman & Goldberg, 1967) contains phenol oxidase in an inactive or proenzyme form. The anterior end of a late third-instar Sarcophaga larva was pricked with a fine pin and the haemolymph released (approx. 5 0 ~ 1 ) was collected in an ice-cold tube. By using this method about 4ml was collected and centrifuged at 2700g for 15min to remove the haemocytes. The cell-free supernatant (plasma) was diluted tenfold with cold distilled water (Price, 1967) and left to stand on ice for 2-3 h during which time a yellow precipitate formed. Occasionally it was found necessary to adjust the pH of the diluted plasma to 6.0 before precipitation occurred. The precipitate was collected by centrifugation (2700g for 15min), washed three times in cold distilled water and dissolved in a small volume (2.0mI) of Ringer's medium, pH6.5 (Price, 1972). This preparation, referred to as the enzyme preparation, is yellow and accounts for approximately 6% of the total plasma protein. Usually the freshly-prepared enzyme preparation possesses a low amount of catecholase activity, but on being left for 24h at 4°C the preparation darkens and activity increases considerably. Phenol oxidase activity is not detectable in the supernatant obtained after the water-dilution treatment. The activity of the preparation is retained for at least 1 week when stored at 4°C. Evans (1967) showed that tenfold dilution of the haemolymph from the Chinese Oak silkmoth, Antheraea pernyi, with distilled water resulted in activation of the phenol oxidase, however, no precipitation was reported. In the present work activity of the preparation was assayed by measuring the O2 uptake at 25°C with a Rank oxygen electrode, the incubation mixture being enzyme preparation (0.05ml) and 3.01111 of 0.01 hi-phosphate buffer, pH6.5, containing 0.01 Mmethyl catechol. Michaelis constants obtained for methyl catechol, dopa (3,4dihydroxyphenylalanine) andp-cresol were 4.5 x 1 0 4 ~ , 4 . 2 ~ 1 0 3 ~ and 1 x 1 0 3 ~ respectively. The ability of the enzyme to oxidize monophenols and diphenols classifies it as a tyrosinase (EC 1.14.18. l), although only low amounts of activity were obtained with tyrosine as substrate, and the addition of small amounts of dopa did not have any catalytic effect.