Abstract
Abstract Size and charge homogeneity and solubility are important parameters of the growth of quality crystals for structure determination of proteins. Horseradish peroxidase isozyme C (HRP) contains 8 heterogeneous N-linked glycans which have hampered crystallographic studies. We have prepared fully active, homogeneous HRP by treatment with trifluoromethanesulfonic acid using a modified protocol and four steps of chromatographic purification. Coprinus cinereus peroxidase (CIP) and site-directed mutants of CIP are secreted in high yields to the fermentation medium of Aspergillus oryzae transformants. Wild-type CIP contains 1 N- and 2 O-linked heterogeneous glycans, which were removed by mutagenesis yielding the ON mutant. In addition, 1 N, 2 N, 4 N and 6 N glyco-mutants were constructed. The enzymatic activities of these mutants were identical to that of the wild-type. Comparison of the properties of the fully glycosylated wild-type HRP and deglycosylated HRP, and of the properties of the wild-type CIP and the glyco-mutants of CIP, showed that the thermodynamic heat and denaturant stability was very little affected, whereas the kinetic stability, i.e. the rates of peroxidase unfolding and refolding, decreased significantly depending on the extent of glycosylation. Increasing contents of carbohydrate greatly increased the solubility in salt solution and decreased the solubility in acetone-water mixtures.
Published Version
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