Observing mutant and normal alkaline phosphatase under varying pH conditions can be used to show the difference between the enzyme and to determine if they function at different pH optima. Patients with moderate kidney dysfunction may be experiencing a reduction in bone density due to the inhibition of alkaline phosphatase and this may be due to the mutation that allows it to function at a different pH optimum (Kirschtel 2019). Through the use of spectrophotometric assay, we determined the absorbance peak and extinction coefficient needed to find the pH optima of the normal and mutant AP enzymes. We learned the pH optimum of the normal AP was approximately 10 and the optimum of the mutant was approximately 7 through the use of an optimal time course. Through the use of SDS-PAGE, we determined that the mutant and normal AP did not just differ in pH optima but also in structure and function. The structural differences and differences in pH optima of the enzymes could account for the reduced bone density that is occurring in patients with moderate kidney dysfunction.
Introduction
Phosphatases are a group of enzymes that can produce a free phosphate ion by cleaving a phosphate group from an organic molecule (Kirschtel 2019). The functions of these enzymes can be altered by a variety of conditions such as pH level, temperature, and salt concentration that the enzyme is exposed to. Metabolic Acidosis is a disorder that occurs due to failure of regulation of acids and bases in the blood (Kirschtel 2019). Long-term metabolic acidosis can result in reduction of calcium hydroxyapatite and loss of bone density (Kirschtel 2019). In vitro studies have suggested that there is a direct correlation between the inhibitory effect of metabolic acidosis on the function of osteoblasts (Disthabanchong et al. 2007).
The colorless substrate, p-nitrophenyl is used as an indicator for Alkaline Phosphatase activity (Bessey et al. 1946). When the phosphate group is split off, the yellow salt of pNPP is set free and thus indicates the measure and presence of AP activity (Bessey et al. 1946). Previous studies have been completed regarding the function of AP and pNPP and have found that the enzyme can undergo conformational changes under varying conditions such as pH level, substrate concentration, buffer, period of incubation, and temperature (Chaudhuri et al. 2013).
In our experiment, we will test whether mutant and normal AP enzymes have different pH optima and whether the enzymes are different in structure. To do this study, we will expose the mutant and normal AP to various pH buffers and measure their activity after a set incubation time. Our hypothesis is that the mutation in the mutant AP allows the enzyme to function at a different pH than the normal AP. This would occur due to the differing structures of the enzyme which would experience different pH optima. Any change in structure of an enzyme will change its function and may have an impact on the overall pH optima.
Methods
All methods in this experiment were taken from (Kirschtel 2019). To begin the experiment, we needed to establish an absorbance spectrum for pNPP to find the absorbance peak and extinction coefficient. To do this, we ran a spectrophotometric assay with a series of five cuvettes, one labeled as the blank. Each cuvette contained varying pNP dilutions. The cuvettes were run in the spectrophotometer at 350 nm and increments of 25 nm until 600 nm. We determined the absorbance peak (398.08 nm) and extinction coefficient (21.48) in order to continue with the experiment and determine the pH optimum of AP.
After finding the absorbance peak and extinction coefficient, we needed to determine the optimal incubation time for the enzymes. In order to do this, we ran a spectrophotometric assay with a series of five cuvettes and determined the optimal time course for incubation was 13 minutes. Using six clean cuvettes, we added 0.25 ml of each pH buffer (6.0, 7.0, 8.0, 9.0, 10 and 11) to separate test tubes. We then added 0.25 ml of pNPP to each cuvette. All six of the cuvettes were then placed in a 37° water bath and left for five minutes. Then in two minute intervals, we added 0.1 ml of enzyme solution to the cuvettes, mixed them, and recorded the time. After the enzyme was added to the cuvettes, the cuvettes were then incubated for 13 minutes. Following their incubation, we added 5.0 ml of 0.05 N NaOH to the cuvettes to terminate the reaction. The cuvettes were then run in a spectrophotometer at 398.08 nm and the absorbances for each pH buffer were recorded.
After the pH optima were determined, an SDS-PAGE was used to determine if there was a difference between the mutant and normal AP. Our TA set up the apparatus and ran the polyacrylamide gels. Each group loaded three samples on a gel of 15 microliters of Normal AP extract, Mixed AP extract, and Mutant AP. The gels were run at 200 volts for 30 minutes. After the gels were run, the TA washed the gels with 50 ml AP Buffer, pH 6. After this was repeated twice, the TA added 50 ml Carbonate Buffer, pH 10 to one tray and 50 ml Citrate Buffer, pH 6 to the second tray. Following this, both trays were stained with 1 ml AP Substrate solution and 1 ml AP Dye solution. The trays were then placed in a 37° Celsius water bath to incubate. Finally, we added 50ml PBS and swirled the trays for two minutes to clean off the excess to be able to sketch the gels and different positions and intensity of the bands.
Results
After completing the absorbance spectrum, we found that the peak absorbance was 398.08 nm and the extinction coefficient was 21.48. The optimal time course that we determined was 13 minutes. Utilizing the optimal time course of 13 minutes, we found that the optimum pH of the normal AP enzyme was approximately 10.0 and the mutant AP was approximately 7.0. The SDS-Page showed that the normal AP functioned best at pH 10 as the bands are the longest and most distinct and intense in the normal AP lane. The mutant AP functions better at pH 6 as seen by the longer more distinct and intense bands seen in the pH 6 tray in the mutant AP lane.
Discussion
Mutant and normal AP were observed under varying pH conditions in order to determine if the two enzymes function at different pH optima and if the two enzymes were different from one another. We hypothesized that the mutation in the abnormal alkaline phosphatase allows the enzyme to function at a different pH than the normal alkaline phosphatase. We predicted that, as a result of the change in structure, the pH optima and overall function of the enzyme would be changed by the mutation. Our hypothesis turned out to be correct. When exposed to a various pH levels, we found that the optimum pH level for normal AP was approximately 10 and the optimum pH level for mutant AP was approximately 7. Due to the difference in the overall structure of the enzymes, which are a major key to their function, this difference in pH optima was not very surprising.
This study shows that the mutation in AP could be the cause of reduced bone density in patients with moderate kidney function (Kirschtel 2019). Previous studies have found that Alkaline Phosphatase has been associated with calcification of the coronary artery in patients suffering from Chronic Kidney Disease (Park et al. 2010). More studies could be done in looking at the direct association between AP activity and reduced bone density as many studies seem to focus more on the association between metabolic acidosis and AP activity. There may be another factor responsible for reduced bone density and metabolic acidosis that we are unaware of. It is crucial to determine the other factor that can lead to metabolic acidosis in patients as it can lead to increased excessive weight loss, fatigue, bone loss, and protein decomposition (Kovacic et al. 2003).
Literature Cited
Bessey, O.A., O.H. Lowry, and M.J. Brock. 1946. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem. 164: 321-329.
Chaudhuri, G., Chatterjee, S., Venu-Babu, P., Ramasamy, K., Thilagaraj, R. (2013). Kinetic behaviour of calf intestinal alkaline phosphatase with pNPP. Indian Journal of Biochemistry & Biophysics. 50. 64-71.
Disthabanchong, S., Radinahamed, P., Stitchantrakul, W., Hongeng, S., & Rajatanavin, R. (2007). Chronic metabolic acidosis alters osteoblast differentiation from human mesenchymal stem cells. Kidney International, 71(3), 201–209. doi: 10.1038/sj.ki.5002035
Escobar-Palermo, P., Gilliam, C., Gross, P., Higgins, W., Kary, C., Keller, M., Kirschtel, D., Lanford, P., O’Brien, T., Spilatro, S., Thompson, K. (2019, Fall/ 2020, Spring). BSCI171 Laboratory Manual: Principles of Molecular and Cellular Biology. College Park, Maryland: University of Maryland College Park
Kovacic, V., L. Roguljic, and V. Kovacic. 2003. Metabolic acidosis of chronically hemodialyzed patients. Am. J. Nephrol. 23: 158-164.