Reactive Thiol Groups in Rat Liver Acid Phosphatase

Abstract

Dimeric rat liver acid phosphatase PI of Mr 92,000 is inactivated by ρ-chloromercuribenzoate and fluorescein mercuriacetate (FMA). The enzyme is protected against the mercurials by the substrate analogue P(i). The reaction with FMA is accompanied by changes in absorbance at 495 nm and in fluorescence emission at 520 nm that are characteristic of reaction of this compound with thiol groups. Titration of PI with FMA monitored by spectrophotometry or by fluorimetry indicated that equivalence is reached at an FMA/P1 ratio of 3. Since FMA can act as a bifunctional reagent, it is likely that PI contains either 3 or 6 reactive thiol groups per molecule. Analysis of FMA inactivation/modification data by a statistical method suggests that of 6 reactive thiol groups, 2 are essential so that there are probably 3 thiol groups per subunit, one of which is located at the active site. If the total thiol number is 3, analysis suggests 1 essential thiol per subunit.