[81] Homogentisate oxygenase (pseudomonas fluorescens)

Publisher Summary This chapter discusses the assay, purification, and properties of homogentisate oxygenase. It can be assayed by two methods—namely, by measuring O 2 consumption manometrically and by measuring 4-maleylacetoacetate spectrophotometrically at 330 m μ . The latter method cannot be used for assaying crude extracts, because they accumulate no maleylacetoacetate. A unit of enzyme activity is the amount of enzyme that consumes 1 micromole of oxygen per minute with the manometric method or produces 1 micromole of maleylacetoacetate per minute with the spectrophotometric method. Specific activity is defined as units of activity per milligram of protein. This enzyme shows maximal activity at about pH 6.0. At pH 6.0–7.2, the enzyme can be stored for several weeks at -20° or several days in a refrigerator without the loss of activity. The enzyme is sensitive to oxygen and easily inactivated by aeration. No activity can be detected in the absence of ferrous ion. This enzyme is inhibited by 85% by 2 × 10 -4 M p -chloromercuribenzoate.