Studies on the roles and identification of the small molecule factors on phenolase activation in Norway lobster ( Nephrops norvegiens)

Abstract

The direct effect of small molecule factor(s) on the activation of phenolase in the suggested multiple component process of blackspot development in Norway lobster ( Nephrops norvegicus) was studied. When acetone-precipitated phenolase was treated with the small molecule filtrate, the phenolase activation profile was lower compared to the control, particularly at pH 6.7. However, when purified form I phenolase was treated with small molecule filtrate no activation was observed, the assayed phenolase activity being constant but lower than the control. A similar effect was observed when form I phenolase was treated with 1.5 mM dihydroxyphenylalanine (DOPA) (approximately the concentration found in the small molecule filtrate), indicating that this lower pattern resulted from the competition of the phenolase substrates (catechol, DOPA, and possibly tyrosine) in the small molecule filtrate. When the activity of the three proteases in the acetone-precipitated phenolase was studied, it was found that there was a high recovery of proteases I and II in the precipitate but little protease III. This also confirms that the effect of the small molecule filtrate on phenolase activity in acetone-precipitated phenolase was due to its indirect effect on the proteases present. The modification of the compounds that absorb at 270 nm was studied by a high-performance liquid chromatographic (HPLC) separation method and the quantitative analysis of tyrosine and DOPA in the small molecule filtrate was achieved. Some of the small molecules were identified, by an HPLC method, as tyrosine, DOPA, oxidised DOPA, vitamin C in oxidised form or breakdown product of vitamin C. Oxidised DOPA, DOPA, tyrosine and N-acetyldopamine inhibit the proteases to varying degrees. Vitamin C also inhibits all three proteases at pH 6.4; however, at pH 8.2 it markedly activates protease III and slightly activates the two thiol proteases (I and II).