1. Introduction
2. Data concerning Alkaline Phosphatase (AP)
   1. Nomenclature
   2. Substrate specificity, relative rates and Km
   3. Enzyme structure and MW
   4. pH optimum
   5. Activators
   6. Inhibitors
   7. Temperature
3. Incubation with Streptavidin-Alkaline Phosphatase conjugate
4. Substrates for AP tested in DIAPOPS
   1. 4-Methylumbelliferyl Phosphate (4-MUP)
      1. Storage of the 4-MUP dilution
   2. para-Nitro Phenyl Phosphate (pNPP)
5. Conclusion

1. Introduction
The alkaline phosphatase enzyme is used in the DIAPOPS procedure to generate a color or fluorescence signal from a substrate ( ). The enzyme has been tested in DIAPOPS either directly labelled to the detection probe ( ) or, most frequently, conjugated to streptavidin that specifically recognizes a biotin group on the probe ( ).

2. Data concerning Alkaline Phosphatase (AP)
The following data concerning AP is a summary of the material provided with the pure enzyme sold by Boehringer Mannheim (Phosphatase, alkaline (AP), EIA grade, Cat. No.: 567 752). This enzyme is used by DAKO A/S to prepare the streptavidin conjugated enzyme (DAKO, Streptavidin-AP, Cat. No. D0396), which is used in the DIAPOPS procedure.

The data describe the unconjugated enzyme. No data have been found on the optimal conditions of the conjugated enzyme. Although it is to be expected that the conjugated enzyme may yield slightly different optimum conditions in practical experiments, no difference has been observed in the numerous DIAPOPS experiments between the data given below and the performance of the streptavidin conjugated enzyme.

2 a). Nomenclature
Alkaline Phosphatase (AP): Orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1.

2 b). Substrate specificity, relative rates and Km
Alkaline phosphatase (AP) hydrolyzes phosphate esters of primary and secondary alcohols, cyclic aliphatic alcohols, sugar alcohols, phenols, and amines. AP also hydrolyzes inorganic pyrophosphate (at a pH optimum of approx. 8) and 5'-terminal phosphates of single and double-stranded DNA or RNA. The enzyme will not hydrolyze phosphodiesters (R-O-PO2-O-R'; R, R': alkyl groups). Representative Michaelis constants (Km determined in 0.1 M Tris, pH 8.0 at 25ºC) include 4-nitrophenyl phosphate, 3.6 µM (relative rate = 1.0); pyrophosphate, 16 µM (rate = 0.9); AMP, 18 µM (rate = 1.2) ATP, 16 µM (rate = 0.9) and phosphoenolpyruvate, 5.5 µM (rate = 0.8).

The hydrolysis rate and substrate affinity of AP depend on a complex interrelation between the purity and the concentration of enzyme, buffer composition, ionic environment (ionic strength, solvents present), and pH. Specific use of AP requires careful, empirical optimization of the reaction conditions.

2 c). Enzyme structure and MW
AP is a dimer (MW = 140.000) of identical or nearly identical sub-units (MW = 69,000), each containing 2 molecules of Zn²⁺, one tightly bound and necessary for structural stability, the other loosely bound and required for catalytic activity. The active site contains a reactive serine. In some mammals (e.g. human), there are at least 3 distinguishable isoenzymes: the intestinal, the placental and the form found in bone/liver/kidney.

2 d). pH optimum
The optimal pH is between 8.0-10.5 (depending on substrate concentration). Figure 1 shows the dependence of pH of the activity under normal assay conditions such as those in the DIAPOPS procedure ( ). The pH optimum of the AP reaction apparently increases with increasing concentrations of substrate. The observed rise in pH optimum is actually due to a rise in both maximum velocity (Vmax) and Km of the enzyme with increasing pH. On Figure 1 is shown the optimum pH at the substrate concentration also used in DIAPOPS. For this reason, the pH in the DIAPOPS enzyme buffer is also 9.8 ( ).

Figure 1: Relative AP enzyme activity as a function of the pH.

2 e). Activators
Activators of AP are: Divalent metal ions (Mg²⁺, Co²⁺, Mn²⁺), amino alcohols (2-amino-2-methyl-1-propanol, diethanolamine), and TRIS buffer. In DIAPOPS, a diethanolamine buffer is used at pH 9.8 ( ). The optimal activity of AP depends on the concentration of Mg²⁺ and Zn²⁺ in the reaction mixture. A low concentration of Zn²⁺ is required for catalytic activity. The Zn²⁺ is bound at a catalytic site. Mg²⁺ acts as an allosteric activator of AP, binding to a site on the enzyme which is distinct from the Zn²⁺ site. However, if present in excess of the amounts necessary for catalysis, Zn²⁺ will also bind to the Mg²⁺ site (with greater affinity than Mg²⁺) and thereby reverse the Mg²⁺ activation effect.

2 f). Inhibitors
Inorganic phosphate (P_i), monoethanolamine, Be²⁺, chelators of divalent metal ions (EDTA, oxalate, citrate, cysteine, histidine), acid or neutral pH, aromatic amino acids (Phe, Trp), L-homo-arginine, urea, iodoacetamide, and high levels of Zn²⁺ are inhibitory.

2 g). Temperature
The temperature dependence of the normal activity and the thermal stability of the enzyme after 10 minutes incubation in 3 M NaCl, pH 7.6, is presented in Figure 2.

Figure 2: Left: Temperature dependence of the activity. Right: Thermal stability after 10 min. incubation.

3. Incubation with streptavidin-alkaline phosphatase conjugate
Incubation with the streptavidin-enzyme conjugates is carried out at room temperature (RT) in standard ELISA procedures. In the DIAPOPS procedure ( ) the incubation temperature is either 37ºC or 50ºC ( ). RT was used in the start of the optimization period of the DIAPOPS procedure as in standard ELISA procedures. However, a very high day-to-day variance was observed in the hybridization results. For this reason incubations at both 37ºC and 50ºC were tested. With both temperatures, results with a much smaller day-to-day variation was achieved. Incubation at 50ºC may be used in the DIAPOPS procedure. When using this temperature, only RT and 50ºC are necessary in the DIAPOPS procedure. The use of this temperature is therefore more convenient as it decreases the need for thermal incubators.

4. Substrates tested in DIAPOPS
Only two substrates for AP have been tested in DIAPOPS, 4-MUP and pNPP. A comparison of the two tested substrates in a DIAPOPS assay is presented in Figure 6 ( ). In this experiment, the same NucleoLink Strips were analyzed twice for solid phase PCR products using rehybridization ( ). In the first detection 4-MUP was used as substrate, and pNPP was used in the second detection. From Figure 6 it is obvious that both substrates can be used, and no difference in performance is observed.

4 a). 4-Methylumbelliferyl Phosphate (4-MUP)
4-MUP is a very sensitive substrate for AP, and it performs satisfactorily in DIAPOPS. The substrate becomes fluorescent when the phosphate group is removed by the hydrolyzation of AP. For detection of hydrolyzed 4-MUP, measure in a fluorescence plate reader. Use an excitation wavelength of 360 nm and an emission wavelength of 450 nm, even if the reaction has been stopped with K2HPO4 ( ). The substrate is used at a concentration of 1 mg/ml in the DIAPOPS procedure, and can be diluted in the enzyme buffer before use and stored in a ready-to-use solution at -20ºC ( ).

Figure 3: 4-MUP structure

4 ai). Storage of the 4-MUP dilution

Figure 4: Showing the difference in fluorescence of four enzyme solutions stored at -20ºC for different amounts of time. The results show, that no decrease in efficiency, and no raised background signals from autohydrolysis are seen, even after a period of 10 months storage.

A comparison was made of the ability to create a signal and of the background level from four different batches of 4-MUP ready-to-use substrate dilutions stored for up to 10 months at -20ºC ( ). The results are presented in Figure 4. In this experiment a dilution of streptavidin-alkaline phosphatase conjugate was made, and the four different batches of substrate dilution was added. The fluorescence was measured after incubation for 15 minutes at room temperature. The highest conjugate concentration is also the dilution used in the DIAPOPS procedure ( ).

The results show, that there is no difference in the ability of the four batches of 4-MUP to be hydrolyzed by the enzyme. Furthermore, no difference in background level is observed, indicating that no autohydrolysis has occured during the 10 months of storage at -20ºC.

4 b). para-Nitro Phenyl Phosphate (pNPP)

pNPP is a substrate normally less sensitive than 4-MUP, but the advantage of this substrate is, that the hydrolyzed product from the enzyme reaction can be measured by light absorbency in a standard ELISA reader. For detection of hydrolyzed pNPP, the OD must be measured in a normal ELISA plate reader at a wavelength of 405 nm ( ). This wavelength is also used when the reaction has been stopped with NaOH ( ). In a standard ELISA assay, the substrate is used at a concentration of 1 mg/ml. However, if this concentration is used it must incubated overnight to yield acceptable results in the DIAPOPS procedure. Faster results may be acquired if the substrate is used in a higher concentration of 10 mg/ml. With this concentration, a result comparable to the result yielded with 4-MUP can be observed after an incubation of only 30 minutes at room temperature ( ). This can be seen in Figure 6. In this experiment, the same NucleoLink Strips were analyzed twice for solid phase PCR products using rehybridization ( ). In the first detection, 4-MUP was used as substrate, and pNPP was used in the second detection. From Figure 6 it is obvious that both substrates can be used, and that no difference in the limit of detection of dynamic range is observed. Normally, the substrate is available in tablets or as a powder.

Figure 5: pNPP structure

Figure 6: A comparison between substrates 4-MUP and pNPP. The same NucleoLink Strips were detected with rehybridization ( ). 4-MUP was used in the normal concentration of 1 mg/ml, but pNPP was used in concentration of 10 mg/ml which is higher than the normal concentration of 1 mg/ml used in standard ELISA. As seen there is no difference in the results. In this experiment, there was no difference in the sensitivity between the two substrates. This indicates that both substrates may be used without any change in performance.

5. Conclusion
Alkaline phosphatase may be used in the DIAPOPS procedure with high performance. The incubation temperature of the Streptavidin-AP conjugate binding incubation should be controlled, and both 37ºC and 50ºC can be used. Two substrates for this enzyme have been tested in the DIAPOPS procedure, 4-MUP and pNPP. 4-MUP is the more sensitive substrate, but, if used in a concentration of 10 mg/ml, pNPP can give results as quickly and with the same performance as that of 4-MUP. Ready-to-use dilutions of 4-MUP in diethanolamine can be stored for up to 10 months at -20ºC without losing the ability to be hydrolyzed and without creating a higher background signal.