Alkaline Phosphatase 335
Incubation with Zn 2+ can reverse some of the inactivation by these
agents, but Mg 2÷ and Co 2÷ are much less effective (29).
Many amino acids are weak inhibitors of CIAP, including L-pheny-
lalanine and L-tryptophan (1, 34), whereas D-phenylalanine has no effect
(31). Inhibition by glycine, cysteine, and histidine is probably owing
to chelation of the zinc ions (35). Diisopropylfluorophosphate, an
inhibitor of other serine hydrolases, has only a slight effect on alkaline
phosphatase (at 1-10 mM) (36). Finally, the enzyme is also inhibited
by high substrate concentrations (millimolar amounts), the precise
mechanism of which is unclear (1).
3.6. Sulfhydryl Reagents
The presence of sulfhydryl reagents is not required for reaction.
Alkaline phosphatase is reversibly dissociated by thiol reagents in the
presence of urea (13).
- Enzyme Assay and Unit Definition
One unit of alkaline phosphatase is defined as that amount of enzyme
that will hydrolyze 1 ~mol of 4-nitrophenyl phosphate/min (37). How-
ever, other conditions can vary with suppliers.
The assay is normally performed in 1M diethanolamine buffer, includ-
ing 10 mM 4-nitrophenyl phosphate and 0.25 mM MgC12. However, a
variety of conditions have been employed resulting in assays at pH 8.0
(12), pH 9.6 (38), pH 9.8 (26,39), or at pH 10.5 (25,40). The assay tem-
perature has also been defined as 37°C (25,26,39,40) or as 25°C (12,38).
The variation in unit definitions is illustrated by the fact that 5 U measured
in Diethanolamine buffer at 37°C are equivalent to 1 U in glycine/NaOH
at 25°C (38), and the presence of 1M diethanolamine in a buffer can
double the observed reaction rate (40). This plethora of unit definitions
illustrates the wisdom of titrating the quantity of alkaline phosphatase
required for any particular operation. A functional unit of activity is some-
times defined as being that quantity of enzyme that will dephosphorylate
1 ~tg (or 1 pmol) of a particular DNA species in 1 h.
- Enzyme Assay and Unit Definition
- Experimental Procedures
4.1. Storage and Stability
The purity of alkaline phosphatase preparations may vary widely,
and this has effects on the stability of the enzyme, as well as contaminating
catalytic activities. Commercial preparations are usually assayed to be