A study on ascorbate inhibition of ceruloplasmin ferroxidase activity

The ferroxidase activity of ceruloplasmin is often determined according to the method of Johnson et al. (1967), using apotransferrin for trapping ferric ions generated by the enzyme; spectrophotometrically monitoring the Fe(3+)-transferrin formation at pH 6.0. Reports have shown that ascorbate inhibits this reaction, and it is hypothesized that the effect could be of physiological significance in individuals with a high ascorbate to ceruloplasmin ratio in plasma (e.g. premature babies). The present study shows that the inhibitory effect of ascorbate rapidly decreases with increasing pH. At pH 7.4 no significant effect was observed, the result suggesting that ascorbate is not a physiological inhibitor of ceruloplasmin. Furthermore, experiments demonstrate that at acidic pH the inhibitory effect of ascorbate on the rate of Fe(3+)-transferrin formation is not primarily due to an interaction with ceruloplasmin, but to a reduction of enzymically generated ferric ions before they are bound to apotransferrin.