Mavicyanin, a Blue Copper Protein from Cucurbita pepo medullosa

Abstract

The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa. Mavicyanin contains one type‐1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (ɛ= 5000 M−1 cm−1/Cu, A280/A600= 8.0 ± 0.5, A600/A403= 7.0 ± 0.25, maximum of fluorescence emission at 335 nm. In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz= 2.287, gy= 2.077, gx= 2.025, Az= 3.5 mT, Ay= 2.9 mT and Ax= 5.7 mT. Like other simple type‐1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicy‐anin is readily reduced by hydroquinone or L‐ascorbic acid. Its midpoint potential E′m was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.

Gene Synthesis, Expression, and Mutagenesis of Zucchini Mavicyanin: The Fourth Ligand of Blue Copper Center Is Gln

“…The midpoint potential is close to the potential (+184 mV) of stellacyanin (25). The midpoint potential of wildtype mavicyanin was reported to be +285 mV (pH 7.0) by reductive titration methods (1). The difference between the midpoint potentials may be due to the carbohydrate moiety.…”

Ascorbate Oxidase from Cucurbita pepo medullosa

“…Whereas for the first group the yield of ascorbate oxidase was highest, it slightly decreased in the larger fruits. For mavicyanin the opposite behavior was observed [22].…”

“…The crude enzyme juice has been shown to contain at least two other metalloproteins in addition to ascorbate oxidase : a peroxidase, which under the conditions of purification is obtained in both a cationic and an anionic form as judged from electrophoretic investigations [38], and a low-molecular-weight protein [22], which contains one single type-1 copper with physical characteristics similar to the copper of stellacyanin [19]. The close location of ascorbate oxidase and peroxidase in plants has already been stressed by Huszak [37], who then assumed that the copper enzyme might play an integral part of the peroxidase system [5].…”

“…The combined fractions (total A610 = 2.27, 1.0-cm cell) were passed through a second DEAEcellulose column, 5.5 x 2.5 cm, and the column was washed with sodium chloride, 0.03 M in 0.01 M sodium phosphate buffer, pH 7.0. By this procedure a small quantity of mavicyanin [22] could be separated from the multicopper enzyme. Finally the second DEAEcellulose column was eluted with 0.1 M sodium phosphate buffer, pH 7.0, and the eluate immediately dialysed against 0.01 M sodium phosphate buffer, pH 7.0, for about 32 h.…”

“…As discussed in [22]. this copper protein exhibits many physical and chemical properties very much alike to those of stellacyanin from the Japanese lacquer tree [19].…”