Javier A. Fuentes BSCI105 Sec.

5015 TA: Jasmine Rajbhandary

Heterozygous traits in the gene responsible for the production of alkaline phosphatase create a mutant enzyme that functions at a lower pH optimum and has a different molecular weight than that of normal alkaline phosphatase. Abstract Alkaline phosphatase is the enzyme responsible for the regulation of the mineral component of bone, hypoxyapetite which is a metabolic pH buffer to the blood. Increased blood acidity can inhibit alkaline phosphatase which can lead to the loss of bone density. Some people happen to be heterozygous to a misfolded mutant AP enzyme which functions at a different pH optimum than that of normal AP. By means of spectrophotometric techniques, I determined the optimum wavelength and developed a standard curve with a specific extinction coefficient. Using an AP specific time course, I learned that the pH optimum of the mutant AP is 6.0 and that of the normal AP is 11.0. This along with an SDS-PAGE of both the homozygous and heterozygous enzymes, show that there are significant differences between the normal and mutant AP. These differences in pH correspond to the differences in folding between the two enzyme types. Introduction Enzymes belong to the class of organic molecules of proteins, which are made of a sequence of amino acids. Each protein is formed based on the translation of mRNA in the ribosomes to the corresponding tRNA into unique sequences. Temperature, pH, solute
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concentration, and salt content are several factors that affect enzyme (and proteins in general) folding. An abnormal protein can differ from a normal protein by simply changing an amino acid in the sequence or its corresponding substituent groups interactions with others along the chain. This small, abnormal folding of proteins can have an extreme impact on the overall biological system of the organism. This is exactly what occurs with the alkaline phosphatase enzyme. The alkaline phosphatase enzyme is a key enzyme in the regulation of deposition of hypoxyapetite by osteoblasts. Hypoxyapetite is used in the blood as a buffer to maintain the pH homeostasis of blood (Arnett 2008). A physiological condition that arises from a failure to control the pH of the blood is metabolic acidosis. This condition can cause an imbalance in mineral homeostasis which leads to the reduction of hypoxyapetite and loss of bone density. It was originally thought that inhibition of the alkaline phosphatase enzyme by high acidity may be a primary cause of reduced bone density in diabetic teenagers with moderate kidney dysfunction. During the analysis of 1,500 samples, it is discovered that certain individuals have a heterozygous trait for the mutation of alkaline phosphatase that cause the enzyme to fold incorrectly (Keller et al. 2012). Along with this, medical literature indicates that the human AP enzyme has an optimum activity at a pH of 10.0. The AP enzyme is functionally abnormal if the enzyme is functioning at a pH other than 10.0 (Keller et al. 2012). Our goal with this experiment is to determine whether or not there is a difference between the mutant AP enzyme and the normal AP enzyme, to include pH optimums and molecular weights. Determining this difference will allow us to understand how it affects those patients with metabolic acidosis. To do this study, I will observe a similar catalytic reaction between the AP enzyme and p-nitrophenyl phosphate (PNP). Research shows that AP removes phosphate groups from molecules (Keller et al. 2012). The product that is formed through this
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reaction will be p-nitrophenol and an organic phosphate and will be what we are measuring to find the rate of activity in AP enzymes. I hypothesize that the mutant AP enzyme will differ in both a pH optimum and molecular weights when compared to the normal AP enzyme. This is because a change in the environment, such as pH, will cause certain substituent groups along the protein sequence of the mutant AP enzyme to alter which will cause a change in structure of the enzyme. A change in the structure of any molecule is equivalent to a change in the molecules function and the environment needed for the molecule to operate. Methods In order to begin studying the pH optimums of both the normal and mutant AP enzymes, I needed to develop an absorbance spectrum for the product, p-nitrophenol which is yellow. To do so, I prepared two spectrophotometer cuvettes and labeled one as the blank. The blank consisted of 0.25mL of PNP (which is colorless), 0.25mL of pH 10 buffer, and 5.0mL of 0.05M NaOH. The other cuvette contained 0.25mL of PNP, 0.25mL of pH 10 buffer, and 5.0mL of nitrophenol. I then ran both cuvettes through the spectrophotometer starting at 400nm and increasing in increments of 25nm (up to 700nm) (Fig. 1a). Once I determined the optimum absorbance to be approximately 400nm, I repeated this technique but with 5nm increments between 390nm and 410nm (Fig. 1b). After confirming the optimum absorbance to be 400nm, I proceeded with creating a standard curve using the blank from above and 5 new cuvettes. In the new cuvettes, the final concentrations of p-nitrophenol were as follows: 0.01mol/mL, 0.02mol/mL, 0.04mol/mL, 0.06mol/mL, and 0.08mol/mL. These new solutions were run

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through the spectrophotometer (now set to 400nm) and gave the standard curve (Fig. 2). The result was an extinction coefficient of 1.92. Using the optimum wavelength (400nm) and the average extinction coefficient of the class (1.5), I progressed to determining a time course that was appropriate for the incubation of the AP enzyme. I began by making a blank solution, which consisted of 0.25mL of PNP, 0.25mL of pH 10 buffer, and 5.0mL of 0.05M NaOH. To a series of 5 clean cuvettes, I added 0.25mL of PNP, 0.25mL of pH 10 buffer, and placed them in a warm water bath set to 37C to incubate for 5min. After the 5min, I quickly added 0.1mL of AP enzyme and returned the cuvettes to the water bath. After another 5min, I took the first cuvette out and added 5.0mL of 0.05M NaOH to neutralize the solution. The above neutralization step was repeated every five minutes to the next cuvette (so the 2nd after 10min total, the 3rd after 15min total, etc.). Once all the samples were neutralized, I measured their absorbance (Fig. 3). Unfortunately at this step, we discovered that the cuvette that was to incubate for 20min had been given 0.2mL of AP enzyme while the 25min cuvette did not receive any AP enzyme, thus showing as little to no absorbance. After discussing the time course as a class, the most efficient incubation time was determined to be 20min. With the incubation time of 20min and the extinction coefficient of 1.5, I was able to begin determining the rate of activity of the normal AP enzyme as a function of pH (I did not determine the rate of activity of the mutant AP enzyme because the class was divided into 2; so one half worked with the normal and the other half worked with the mutant.). Obtaining six cuvettes, I added 0.25mL of each pH buffer (6.0, 7.0, 8.0, 9.0, 10.0, 11.0) to separate test tubes (Keller et al. 2012). I then added 0.25mL of PNP to each cuvette and placed all of the tubes in the warm water bath set to 37C. After 5min, I added 0.1mL of normal AP enzyme to the first cuvette and recorded the time (this will run for 20min with the enzyme). The rest of the cuvettes
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would have the AP enzyme added in periods of 2min to allow for the previous cuvette to be taken out of the bath and neutralized with 5.0mL of 0.05M NaOH. Once the incubation was done and all of the samples were neutralized, I proceeded to measuring absorbances, calculating concentrations, and the rates as a function of time (Table 1a). Once the class finished the calculations, class mean rates were calculated (Table 1a and 1b). Using this information on class mean rates, we were able to calculate the pH optimum of both the normal and mutant AP enzymes. An SDS-PAGE was to be done during the experiment to determine if there was a difference between the mutant and normal AP enzymes. The TA set up the polyacrylamide gels and PAGE apparatus, demonstrating how to operate and run a gel through the PAGE apparatus by running a standard gel. Each group loaded 3 samples: (a) 15L Homozygous normal AP, (b) 15L Heterozygous AP, (c) 15L AP mutant enzyme. The TA ran the gel for approximately 20min before cut out the gels and passing them out to each group in staining trays. We added 50mL of AP Buffer, pH 7, and swirled gently for 2min to remove the presence of SDS. We then added approximately 50mL of AP Buffer, pH 6, 1mL of AP Substrate Solution, and 1mL of AP Dye Solution and swirled to mix. The staining tray was then added to the warm water bath (37C) and incubated for 10min with occasional swirling. The above staining process was done 2 more times afterwards for a total of 30min incubating. Afterwards, we added approximately 50mL of PBS and swirled for 2min to rinse the gel. This process should give clearly distinct bands along the gel. Results

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Through completion of the study on the absorbance spectrum of the product pnitrophenol, I observed that the optimum absorbance wavelength is 400nm (Fig. 1a/1b). This optimum absorbance wavelength was used to calculate a standard curve to which an extinction coefficient could be found for p-nitrophenol. I determined this extinction coefficient to be approximately 1.92, and the class average extinction coefficient was determined to be 1.5 (Fig. 2). Utilizing the extinction coefficient and optimum wavelength, a time course was determined for incubation of the AP enzyme (Fig. 3). The time course for my group, unfortunately, faced human error in the fact that the tube that was to be incubated for a total 20min received 0.2mL of AP enzyme while the tube that was to be incubated for 25min had no AP enzyme added. An average time course of 20min of incubation time was determined and used, along with the average extinction coefficient of 1.5, to find the mean rates of activity in both normal and mutant AP enzymes as a function of pH (Table 1a/1b). Using this information, I observed that the optimum pH of the normal AP enzyme was approximately 11.0, while that of the mutant AP enzyme was approximately 6.0 (Fig. 4). Unfortunately, due to complications with the SDS-PAGE equipment or with enzyme samples, we were unable to develop a gel with sufficiently distinct bands (Fig. 5). However, the TA was able to retrieve an old sample to show the class what the bands would have looked like. According to the old sample, the mutant AP enzyme does have a different molecular weight and size which correlates with the difference in folding due to functioning at a much lower pH than normal. Discussion

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A heterozygous gene which produces a misfolded mutant AP enzyme does function at a different pH optimum and has a different molecular weight than that of the normal homozygous AP enzyme. This makes physical, biological, and chemical sense because the change in folding that is present in the mutant enzyme is caused by a change in the pH conditions. This is important to note because there are at least 3 genes that code for AP in humans that each function at different pH optimums and all have different molecular weights (Weiss et al. 1988). This information is extremely crucial for those who have metabolic acidosis, which has adverse effects including protein breakdown, excessive weight loss, fatigue, and bone loss (Kovacic et al. 2003). In the future I would like to test whether differences in substrate will influence the rate of activity of the AP enzyme at its pH optimum. Literature Cited Arnett, T.R. 2008. Extracellular pH regulates bone cell function. J. Nutr. 138: 415S-418S. Keller, Michael J. and Lanford, P.J. 2012. BSCI 105 Introduction to Experimental Biology. Hayden-McNeil Publishing: Plymouth, MI. Kovacic, V., L. Roguljic, and V. Kovacic. 2003. Metabolic acidosis of chronically hemodialyzed patients. Am. J. Nephrol. 23: 158-164. Weiss, M.J., K. Ray, P.S. Henthorn, B. Lamb, T. Kadesch, and H. Harris. 1988. Structure of the human liver/bone/kidney alkaline phosphatase gene. J. Biol. Chem. 263: 12002-12010.

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