Purification and partial characterisation of phenoloxidase from the colonial ascidian Botryllus schlosseri |
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Authors: | A Frizzo L Guidolin L Ballarin A Sabbadin |
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Institution: | (1) Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, I-35121 Padova, Italy, IT |
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Abstract: | Phenoloxidase (PO) from the colonial ascidian Botryllus schlosseri was purified using two different chromatographic strategies. A three-step purification was developed in order to maintain
enzyme activity, whereas an easier purification procedure was adopted to obtain enough PO for the production of specific polyclonal
antibodies. The enzyme showed optimal pH and temperature values of 7.0 to 7.5 and 35 °C, respectively, and a K
m value of 4.62 ± 0.76 mM was estimated using l-DOPA as substrate. A molecular weight of 160 kDa was determined after SDS-PAGE under non-reducing conditions. The addition
of the reducing agent β-mercaptoethanol caused the disappearance of the 160 kDa band and the appearance of a new band at 80 kDa,
suggesting that active PO is a dimer and the two subunits are linked by disulphide bridges.
Received: 14 December 1998 / Accepted: 24 August 1999 |
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Keywords: | |
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