Purification of Aldolase Lab Report

Brian Spearman Partner: Darla Fink

Due: November 6, 2013 Submitted: November 6, 2013

bps5082

2 of 12

MATERIALS AND METHODS: Note: All procedures were performed at 0-4C, and aliquots and fractions were stored at -20C. Extraction An 80.15 g sample of ground rabbit muscle was obtained. The protein content of this sample was extracted by placing it into a solution of 150 mL of 0.03 M KOH and 0.001 M EDTA. This solution was stirred by hand for 20 minutes. This mixture was then centrifuged at 10,000 x g for 10 minutes. The supernatant containing aldolase amongst other proteins was passed through glass wool in order to remove lipids from the solution. The final volume of the Fraction I solution was measured to be 168 mL. A 1 mL aliquot was taken, labeled as Aliquot I, and stored. Salt Fractionation A 168 mL sample of 100% saturated ammonium sulfate solution was added to Fraction I in order to create a 50% saturated solution. This solution was slowly stirred on ice for one hour, and then centrifuged at 10,000 x g for 30 minutes. The supernatant was again filtered through glass wool. The final volume of this Fraction II solution was measured to be 320 mL. A 1 mL aliquot was taken from Fraction II, labeled as Aliquot II, and stored. This solution was then brought to 60% ammonium sulfate saturation by adding 20.8 g of solid (NH4)2SO4 at a very slow rate. The pH of this solution was measured to be 7.0, and so the solution was adjusted with 1.0 N NH4OH to a pH of 7.5. It was stored in the cold room for one week. Dialysis Fraction II was centrifuged at 10,000 x g for 30 minutes. 378 mL of the supernatant was removed without disturbing the pellet, and the pH of this Fraction III solution was measured as 7.7. A 1 mL aliquot was removed from Fraction III and labeled as Aliquot III. Each precipitate

bps5082

3 of 12

was dissolved using 1 mL of cold equilibration buffer (10 mM Tris HCl, pH 7.5, 5 mM EDTA), creating a total volume of 2 mL. This solution was then centrifuged at 10,000 x g and 4C for 15 minutes. The supernatant of this Fraction IV solution was removed and measured to be 8 mL. An aliquot of 0.1 mL was removed from this solution, diluted to 1 mL with equilibration buffer, and labeled as Aliquot IV. A dialysis tube with a 14,000 Da molecular weight cut off was then prepared. Fraction IV was placed inside the tube, and then the tube was dialyzed against equilibration buffer for one week in the cold room. Affinity Chromatography A phosphocellulose column with dimensions 1.5 cm x 11 cm and 20 ml total volume was prepared. Fraction V was removed from the dialysis tubing and centrifuged at 10,000 x g and 4C for 15 minutes. A 0.1 mL aliquot was removed from the supernatant and diluted to 1 mL with equilibration buffer. Fraction V was then loaded into the column and washed using equilibration buffer. This wash was collected until the absorbance of the effluent at 280 nm fell below 0.1 Abs. A 1 mL aliquot was taken from the collected wash and labeled wash. The sample was then eluted using substrate elution buffer (2.5 mM FBP [B-grade]; 50 mM Tris-HCl, pH 7.5; 5 mM EDTA). Fractions 1 mL in volume were collected every minute for 40 minutes, and each fractions absorbance was measured at 280 nm. Fraction 11 was identified as the peak because its absorbance of 2.59 was the largest of any of the fractions collected, and it was therefore determined to have the highest concentration of aldolase. Fractions 10-17 were pooled into Fraction VIA, and Fractions 18-30 were pooled into VIB. They had volumes of 3.5 mL and 7.5 mL, respectively. 0.1 mL aliquots were removed from each fraction, diluted to 1 mL with equilibration buffer, and labeled Aliquot VIA and VIB.

bps5082 Protein Content Determination

4 of 12

The protein content was determined from the aliquot samples from each fraction. A standard curve was created by measuring the absorbance of different concentrations of bovine serum albumin (BSA) at 595 nm. Aliquots I, II, III, IV, V, wash, peak, VIA, and VIB were diluted so that their absorbance at 595 nm fell within the range of the standard curve. Aldolase Assay The following components were added to cuvettes: dH2O, Histidine-HCl buffer (86.67 mM), NaH2AsO4 (12 mM), NAD+ (3.8 mM), FBP (6.67 mM), Ga3P dehydrogenase (400 IU/mL), and the aldolase fraction diluted with dH2O. The absorbance of each cuvette was measured at 340 nm. Triose Phosphate Isomerase (TPI) Assay The following components were added to cuvettes: dH2O, Histidine-HCl (86.67 mM), NADH (0.13 mM), Ga3P (0.1667 mM), -GP dehydrogenase (~40-50 IU/mL), and the aldolase fraction diluted with dH2O. The absorbance of each cuvette was measured at 340 nm. RESULTS: Extraction The absorbance of Fraction I at 340 nm was measured to be 0.106. This correlates to a protein concentration of 0.010179 mg/mL. Salt Fractionation Fraction II had an absorbance value of 0.110 at 340 nm, which correlates to a protein concentration of 0.0014838 mg/mL.

bps5082 Dialysis

5 of 12

The volume of Fraction III was 378 mL, and it had a pH of 7.70. Fraction III had an absorbance value of 0.045, which correlates to a protein concentration of 0.0071688 mg/mL. Fraction IV had an absorbance value of 0.154, which correlates to a protein concentration of 0.013903 mg/mL. Fraction V had an absorbance value of 0.156, which correlates to a protein concentration of 0.012253 mg/mL. Affinity Chromatography The fraction containing the wash had an absorbance value of 0.001, which correlates to a protein concentration of 9.2*10-3 mg/mL. Each collected fraction was 1 mL in volume. Fraction 11 had the highest absorbance and was labeled the peak fraction. The peak had an absorbance value of 0.049, which correlates to a protein concentration of 0.012564 mg/mL. Fractions 10-17 were pooled into Fraction VIA, which had a total volume of 3.5 mL. Fraction VIA had an absorbance value of 0.032, which correlates to a protein concentration of 0.023058 mg/mL. Fractions 18-30 were pooled into Fraction VIB, which had a total volume of 7.5 mL. Fraction VIB had an absorbance value of 0.020, which correlates to a protein concentration of 3.5*10-3 mg/mL. The absorbance of each eluted fraction is found in Figure 1.

Absorbance vs. Fraction Number

Absorbance at 280 nm 3 2.5 2 1.5 1 0.5 0 0 10 20 30 Fraction Number 40 50

Figure 1: Fractions 1 mL in volume were collected every minute from the affinity chromatography column. The absorbance of each fraction was read at 280 nm.

bps5082

6 of 12

In order to determine the experimental protein concentrations in each fraction from absorbance values, a bovine serum albumin (BSA) standard curve was required. This standard curve is found in Figure 2.

BSA Standard Curve Absorbance

0.6 0.5 Absorbance 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 y = -0.0004x2 + 0.0291x - 0.0003

Bovine Serum Albumin Concentration (g/mL) Figure 2: A standard curve was produced by reading the absorbance of the following concentrations of bovine serum albumin at 595 nm: 1, 5, 10, 15, 20, and 25 g/mL. Each fraction was diluted before its absorbance at 340 nm was measured. For each fraction, the dilution factor, its absorbance, and its associated concentration and reaction velocity is recorded in Table 1. Table 1: The dilution factor, Bradford absorbance, protein concentration, and tube velocity for each Fraction. Bradford Tube Protein Tube Reaction Fraction Dilution (fold) Absorbance (g/tube) Velocity (units) 0.255 10.179 0.1224 I 5000 0.042 1.4838 0.02016 II 5000 0.188 7.1688 0.09024 III 500 0.328 13.903 0.15744 IV 4000 0.297 12.253 0.14256 V 6000 0.234 9.2006 0.11232 Wash 200 0.303 12.564 0.14544 Peak 2000 0.461 23.058 0.22128 VIA 3000 0.098 3.5492 0.04704 VIB 200

bps5082

7 of 12

Aldolase Assay The Aldolase Assay was used to determine the activity of aldolase for each fraction. This allowed for the calculation of related values. The data from this assay is summarized in Table 2. Table 2: Data table summarizing all associated calculations for each fraction during the Aldolase Assay.
Fraction I II III IV V Wash Peak VIA VIB Volume (mL) 168 320 378 8 11.5 62 1 3.5 7.5 Activity (Units) 25440 26400 1080 29568 44928 9.6 4704 4608 192 Total Units 4273920 8448000 408240 236544 516672 595.2 4704 16128 1440 Protein (mg/mL) 0.010179 0.0014838 0.0071688 0.013903 0.012253 0.0092006 0.012564 0.023058 0.0035492 Total Protein (mg) 8550.36 2374.08 1354.9032 444.896 845.457 114.08744 25.128 242.109 5.3238 Specific Activity 499.8526 3558.431 301.3057 531.6838 611.1156 5.217051 187.2015 66.61462 270.4835 Yield 100 197.663 9.5519 5.5346 12.0889 0.01393 0.11006 0.37736 0.03369 Fold Purification 1 7.118960 0.602789 1.063681 1.222592 0.010437 0.374513 0.133269 0.541126

Triose Phosphate Isomerase Assay The purpose of the Triose Phosphate Isomerase (TPI) Assay was to determine whether other proteins such as TPI co-purified with aldolase throughout the experiment. The data from this assay is summarized in Table 3. Table 3: Data table summarizing all associated calculations for each fraction during the Triose Phosphate Isomerase Assay.
Fraction I II III IV V Wash Peak VIA VIB Volume 168 320 378 8 11.5 62 1 3.5 7.5 Activity 68160 51120 4728 9408 6048 307.2 96 288 0 Total Units 11450880 16358400 1787184 75264 69552 19046.4 96 1008 0 Protein (mg/mL) 0.010179 0.0014838 0.0071688 0.013903 0.012253 0.0092006 0.012564 0.023058 0.0035492 Protein (mg) 8550.36 2374.08 1354.9032 444.896 845.457 114.08744 25.128 242.109 5.3238 Specific Activity 1339.228 6890.416 1319.049 169.1721 82.2656 166.946 3.82044 4.16341 0 Yield 100 142.857 15.6074 0.65728 0.60739 0.16633 0.000838 0.008802 0 Fold Purification 1 5.14507 0.98493 0.12632 0.06143 0.12466 0.00285 0.00311 0

bps5082

8 of 12

Sample calculations for the data found in Tables 2 and 3 can be seen below as performed for Fraction I in the Aldolase Assay:

A histogram was used in order to best visualize the trends in specific activity of each fraction as calculated through the aldolase and TPI assays. The data for both of these two assays were superimposed together in Figure 3.

bps5082

9 of 12

Specific Activity vs. Aldolase and TPI Assay Fractions

8000 Specific Activity (Units/mg) 7000 6000 5000 4000 3000 2000 1000 0 I II III IV V Wash Peak VIA VIB Assay Fraction Aldolase Assay TPI Assay

Figure 3: The calculated specific activity for each fraction during the Aldolase Assay and the TPI Assay was plotted. DISCUSSION: The first technique used in isolating aldolase was extracting the cell contents from rabbit muscle with osmotic lysis using 0.03 M KOH, 0.001 M EDTA solution. After centrifugation of this solution, the supernatant was passed through glass wool, which removed the lipids. Attempts were made to stabilize the proteins using EDTA to inhibit proteases as well as cold temperatures to avoid disrupting the protein folding. The next step was salt fractionation using ammonium sulfate. When salt is in solution with proteins, the salt ions will compete with proteins for interactions with water. This forces the proteins to instead interact with each other, causing them to precipitate, or salt out. Since different proteins possess different levels of hydrophobicity, different proteins will begin to precipitate at different salt concentrations. This was utilized by first removing any proteins that precipitated at 50% ammonium sulfate saturation. The ammonium sulfate saturation was then increased to 60% so that only proteins that salted out at between 50% and 60% ammonium sulfate saturation would be present in the precipitate. Aldolase is known to salt out at this concentration. Dialysis was then used to

bps5082

10 of 12

remove from solution the ammonium sulfate that was necessary for this process. The final technique used to concentrate aldolase was ion exchange chromatography using a column containing phosphoceullulose resin. Phosphate groups in the resin bind to the Arg-148 residue in aldolase. This keeps aldolase bound to the column while other proteins are eluted. Aldolases natural substrate FBP is then added to the column, causing it to elute from the column.1 There were a few general trends seen throughout this experiment. The total amount of protein in milligrams decreased from 8550.36 mg in Fraction I to 242.109 mg in Fraction VIA and 5.32 mg in Fraction VIB. Likewise, the total aldolase activity decreased throughout the experiment from 4.27*106 units of activity in Fraction I to 1.61*104 units in Fraction VIA and 1.44*103 units in Fraction VIB. Finally, specific activity in units per milligram of protein also exhibited an overall decrease from 499.9 units/mg in Fraction I to 66.6 units/mg in Fraction VIA and 270.5 units/mg in Fraction VIB. It is difficult to determine an overall trend in specific activity because its levels shifted upward or downward depending on the step of the experiment. While each of these three trends was mostly clear, Fraction II consistently contrasted each of the downward trends. These measurements may be explained by the presence of condensation on the cuvette while recording its absorbance and concentration. Each step of the procedure for this experiment was designed to isolate and increase the concentration of aldolase in solution. However, the most effective step in increasing the specific activity of aldolase occurred during salt fractionation, in which a 7.12-fold purification occurred. This is because the process was able to remove much of the non-aldolase protein mass. A side effect of this purification process is that some of the targeted protein is also eliminated, and the step in which the largest loss of aldolase occurred was ion exchange chromatography. This process saw the activity yield decrease from 12.1% of the original protein activity to just 0.11%

bps5082

11 of 12

in the peak fraction and 0.014% in the wash. The purpose of the wash step during the ion exchange chromatography was to elute every protein excluding aldolase, which would remain bound to the column until FBP was added. However, it was observed that much of the aldolase content was lost during this step where it most likely remained in the column. Triose Phosphate Isomerase (TPI) is another common enzyme found in rabbit muscle. The TPI assay was used to determine if other enzymes such as TPI were present after the purification process of the experiment had been completed. The total TPI activity throughout the experiment exhibited a negative trend in which its levels decreased from 1.15*107 units in Fraction I to 1007 units in Fraction VIA and 0 units in Fraction VIB. Based on this observation, it can be concluded that the purification process was successful in eliminating much of the TPI in the original sample of rabbit muscle. When Penhoet and Rutter conducted this experiment, they saw a yield percent of 46 from rabbit liver and 21 from rabbit brain.2 Their yields are several times larger than the 0.377% yield that was observed in Fraction VIA of this experiment. By Fraction III, 90% of the yield was lost, indicating that one significant explanation for the loss occurred during the salt fractionation technique. Aldolase is most stable at a pH of 7.5, but Fraction II had a pH of 7.0 and Fraction III had a pH of 7.7. These environments may have resulted in the denaturation of much of the experiments yield. Although this experiment successfully resulted in the purification of some aldolase, the experimentally observed yield rates were less than ideal. In order to increase these yields, several improvements could be made. The experiment should be held at a constant pH of 7.5 to reduce aldolase denaturing. A longer column could also be used in order to achieve better isolation of aldolase.

bps5082 REFERENCES:
1 2

12 of 12

MacDonald et al, Archives of Biochemistry and Biophysics, 408: 279-285 (2002). Penhoet, E.E. and Rutter, W.J., Methods in Enzymology, 42: 240-249 (1975).