Abstract:
Extracts of viscera of the New Zealand paua, Haliotis iris, were found to hydrolyse nitrocatechol sulphate (2-hydroxy-5-nitrophenyl sulphate), phenolphthalein disulphate, p-nitrophenyl phosphate and a wide range of p-nitrophenyl glycosides. p-Nitrophenyl-β-L-fucose was not hydrolysed.
Multiple forms of acid phosphatase, arylsulphatase and β-glucuronidase were shown to be present in visceral extracts. Forms of arylsulphatase having isoelectric points ~5.O hydrolysed ascorbic acid 2-sulphate. A form with isoelectric point ~7.6 lacked this ability.
An arylsulphatase of isoelectric point ~5.0 was substantially purified by a combination of acid precipitation, ammonium sulphate fractionation, cellulose phosphate chromatography and ConA-Sepharose chromatography. The final preparation contained two very similar enzymes. It is suggested that these represent the dimer (70,O00 daltons) and trimer (105,000 daltons) forms of the enzyme. The enzyme resembled mammalian arylsulphatase A enzymes more than mammalian B enzymes.
The arylsulphatase hydrolysed a variety of substituted hydroxyphenyl sulphates at pH 6.0. Nitrocatechol sulphate displayed substrate inhibition which was relieved by an increase in the concentration of the acetate buffer used. A model, based on competition between substrate and buffer ions for an inhibitory binding site, was developed to explain this effect. Although qualitative prediction was good, quantitative agreement was not extremely close.
The pH optima of hydrolysis and the reaction velocity with various arylsulphatase substrates were strongly dependent on the buffer used. The Vmax values for a variety of substituted hydroxyphenyl sulphates indicate that the rate-limiting step for hydrolysis is neither the breakdown of a sulphate-enzyne complex, nor the breakdown of a phenol-enzyme complex. Sulphate inhibition of nitrocatechol sulphate hydrolysis was neither classically competitive nor classically non-competitive.
Two continuous spectrophotometric assays of arylsulphatase were developed, based on the chelation of copper (II) ions by nitrocatechol. Substrates were nitrocatechol sulphate and 2-hydroxy-4-nitrophenyl sulphate. Both assays had good sensitivity and there was good agreement with alkali-terminatied assays. A reaction lag-phase was evident.
