Fluorescence intensities of naphthol AS-MX were measured under various conditions over an emission range of 400–600 nm in order to show the feasibility of using naphthol AS-MX phosphate for detecting phosphatase activity in non-aqueous environments. Specifically, 5 aprotic: protic ratios (70:30, 75:25, 80:20, 85:15, and 90:10 of 1,4-dioxane: Tris buffer) were tested, with the protic component prepared as 0.1 M Tris at one of two different pHs (7.0 or 8.0).
It is known that the maximum fluorescence intensity (λmax) of naphthol AS-MX in largely aqueous solutions is 512 nm when excited at 388 nm. As illustrated in figure 1, the λmax is indeed within the range of 500 and 550 nm for all conditions with well-defined maxima. For each set of data that resulted from using the same buffer, the λmax value changed as a function of aprotic: protic solvent ratio, with a redshift occurring at higher amounts of aprotic solvent (1,4-dioxane). Interestingly, it has been previously identified that in aqueous solutions, naphthalen-2-ols have a fluorescence that is redshifted upon deprotonation, yet here λmax values are near identical across the pH range tested with the largest changes occurring in higher dioxane percentages. pKa's of compounds, however, have been known to increase with the addition of non-aqueous solvents, and thus this effect may be playing a role in fluorescence intensity.
Naphthol AS-MX has the highest fluorescence intensity in 70% dioxane with the 30% Tris buffer protic component composed using Tris buffer at pH 8.0 (with a λmax value at approximately 515 nm). Another noteworthy finding revealed that as the percentage of dioxane increased, the fluorescence decreased in intensity (with a less defined blueshifted λmax). Specifically, for both Tris buffer pHs used, naphthol AS-MX had the lowest intensity in mixtures corresponding to samples containing 90% dioxane. This decrease in intensity, however, did not appear to be linearly dependent on the aprotic: protic (dioxane: Tris buffer) ratio and larger differences can be at lower dioxane amounts.
As would be expected different buffers used as the protic component of the solvent mix affects results substantially, thus the aqueous buffers used in the present study contained the same amount of the same buffer system (0.10 M Tris(hydroxymethyl)aminomethane). When comparing all of the results, the highest intensity values (emission at 512 nm) corresponded to the more alkaline protic component (i.e., pH 8.0) across each individual solvent ratio. Fluorescence changes from pH differences in the protic component of the solvent may be directly due to protonation state of the compound itself (naphthol AS-MX’s hydroxyl pKa is 7.021) or to changing interactions with the solvent. Thus, additional studies are necessary to further delineate the exact contributions to the observed changes.
Fluorescence of phosphorylated naphthol AS-MX was also investigated under each condition. The results from these experiments are tabulated in supplementary table 1. Importantly, two-tailed Student’s t-tests between naphthol AS-MX and naphthol AS-MX phosphate indicate clear fluorescence differential for all conditions at emission wavelengths between 500 and 600 nm (p-values below 0.05). Also, one-sample, two-tailed Student’s t-tests indicate that naphthol AS-MX phosphate did not fluoresce at all when excited at 388 nm (p-values were above 0.6 for all conditions). Therefore, any detectable fluorescence emission in a reaction starting with naphthol AS-MX phosphate will indicate dephosphorylation to naphthol AS-MX.
Previously, soluble phosphatases have never been shown to have activity in solutions made up of more than 25% organic solvent. Figure 1C, however, shows that in this study wheat germ acid phosphatase (WGAP) was able to convert naphthol AS-MX phosphate into naphthol AS-MX after 3 h in a 64:36 dioxane: aqueous Tris (pH 7.0) mixture, as indicated by fluorescence emission with a maximum around 515 nm (when excited at 388 nm) only when WGAP was added to the naphthol AS-MX. This emission intensity is much lower here than is seen in (Fig. 1) panels A-B due to the use of a lower concentration of naphthol AS-MX and a different fluorimeter. Despite this, p-values of less than 0.05 when using a two-samples of unequal variance, two-tailed Student’s t‑test to compare the samples with or without wheat germ acid phosphatase clearly indicate activity. These data, therefore, display proof of principle that the methodology described in this paper can be used for detection of phosphatase activity in high percentages of organic media.