Tiie Journal of Biological Chemistry

A new procedure has been described for the isolation and purification of ascorbate oxidase from green zucchini squash (Cucurbita pepo meduttosa). The new purification method achieves a more than ZOO-fold purification with about a 14% recovery of the total activity present in the crude juice. The purified enzyme preparations have been found to be homogeneous by the criteria of ultracentrifugal analysis and electrophoresis on polyacrylamide gels. The sedimentation constant has been found to be &,, = 7.52 S and a molecular weight of 140,000 has been estimated. The preparations exhibit a specific activity in the range of 3800 to 4250 units per mg, and contain 0.46 to 0.52% copper corresponding to 10 to 12 atoms of copper per enzyme molecule. These specific activity and copper content values are significantly higher than those of homogeneous ascorbate oxidase preparations previously described. The amino acid composition of the new ascorbate oxidase preparation is similar to that of an earlier homogeneous preparation of the same molecular weight but having a significantly lower copper content and specific activity. The marked increase in specific activity of the new preparation appears to be due to the higher content of prosthetic copper rather than to a difference in the protein moiety.