Physical and Chemical Properties of Aldolase Lismari Reyes Munoz Partner: Aubree Himes Due Date: December 6, 2013

Date Submitted: December 6, 2013

Abstract: In order to characterize aldolase, many steps had to be taken into consideration for the purification and analysis of the protein. Purification of a protein needed various steps such as: dialysis, ammonium sulfate precipitation, and ion exchange chromatography to ensure that aldolase was the only protein present in the solution. Other methods were used to prove the purity of aldolase and observe if aldolase was effectively present in solution without any protein impurities. These methods were TLC, HPLC, crystallization, and IEF and they were used to characterize aldolase in terms of structure and functionality. Easy and reliable methods used to obtain information about small characteristics of the aldolase in short amount of time. The size and molecular weight were determined for different situations of aldolase in its native and acid-treated manner. The crystallization after treating the protein with different kinds of salts, NaCl and NaI, at different concentrations was useful to compare the characteristics in crystal formation. The terminal residues for aldolase were found by using the C-terminal residue analysis. Introduction: Various methods are commonly used to separate and purify proteins and many other compounds. A rabbit aldolase was the protein used in this experiment. This is considered a protein that helps break down certain sugars into energy and that performs an aldol reaction. Some characteristics that are known about the aldolase was the composition of its 363 amino acids and the molecular weight subunit of 39,211. In the native conditions of aldolase, the proteins tend to get very close to one another to ensure stability and also to prevent the hydrophobic regions to be exposed. The rabbit muscle aldolase was isolated from a sample of raw muscle and the goal was to isolate the aldolase to a great extent so that it was the only protein present in the solution at the end. In order to isolate the aldolase, a few techniques had to be used. The first technique was ammonium sulfate precipitation, in which the basic idea was to concentrate and precipitate the aldolase out of the sample solution to leave other proteins or compounds back in the solution. The other separation technique was dialysis, which facilitates the removal of small and unwanted compounds from a solution by selective or passive diffusion through a semi-permeable membrane. In this case, the unwanted molecules diffuse through the membrane into a second chamber of liquid or dialysate. Large molecules were not able to pass through the pores of the membrane meaning that aldolase stayed inside dialysis tube. The aldolase at this point was further purified and stabilized. The ion exchange chromatography was used to dissolve the proteins in a mobile phase fluid and passed through a stationary phase (solid structure) to be able to observe how the proteins in mobile phase were divided into different bands based on the different rates of mobility and molecular weight. This was able to isolate the

aldolase protein even more and remove any other constituents that could have been still present in solution. The SDS-PAGE electrophoresis was then used to make sure that aldolase was the only protein remaining in the solution to be able to obtain the molecular weight and isoelectric focusing for the aldolase residue. The main objective for this experiment was to identify, quantify, and determine the molecular weight of aldolase after a few purification processes at different conditions. Also, identify the terminal residues, crystal formation at different concentration of salts at different pH values, and understand the native and acid-treated conditions at which the aldolase can be found. This was going to be useful to perform further analysis of TLC and SDS-PAGE, and be completely aware on how the purification process works. Material and Methods: SDS Polyacrylamide Gel Electrophoresis (SDS-Page) In this experiment, the SDS-Polyacrylamide gel electrophoresis contains two different types of gels. These two gels needed to be created to aid in the identification of the isolated fractions. Both gels contained a different combination of reagents and a percentage concentration of acrylamide. The percentage of acrylamide in the stacking gel was 4% and 1.5M Tris-HCl at a pH of 6.8, and the separating gel had a percentage of 12% and 1.5M Tris-HCl at a pH of 8.8. In order to prepare the running samples, a gel apparatus was placed in a buffer chamber and filled with part of the 400 ml of 1x electrophoresis running buffer. The rest of the buffer was then added to the outer chamber so that at least the bottom 1 cm of the gel was covered and all the air bubbles had to be removed so that the mobile phase was not affected. Each of the fractions from the previous experiment was diluted with distillated water to obtain a final concentration of 0.5 mg/ml.

When the gels were settle individually and the stacking gel has the comb over to create all the wells, the sample fractions were added. From the dilutions of Fraction I-V, only 16 l was used to be mixed with 24 l 2x SDS sample loading buffer and from the rest of the Fraction VIA, VIB, peak, and water only 8 l was used to be mixed with 32 l of the same buffer. The samples had to be heated at 95C for 4 minutes to disrupt ionic and hydrogen bonds, and centrifuged for just 5 seconds. On each of the wells only 20 l was loaded for each of the samples and only 10 l for the ladder. Samples were added to gently to prevent the wells from flooding over into one another. A molecular weight standard was added to a single well as a standard for checking the size of each wells content. The buffer chamber was run at 200 volts for 45 to 60 minutes until the tracking dye reached the bottom of the gel. The tracking gel, Standard Coomassie Brilliant Blue R-250, which was added to the gel, was tracked to the bottom of the gel, at which the point of charge was remove when the run was

completed. The process took approximately one hour. The gel was then removed and analyzed based on the standard (ladder) that was inserted during the addition of the other sample fractions. High Performance Liquid Chromatography (HPLC) The High performance liquid chromatography (HPLC) or high pressure liquid chromatography was another technique utilized to assess the purity of the peak. The instrumentation used for this kind of chromatography was SSI 210 Guardian HPLC having a column of Progel-TSK G3000 SW (MW range 10,000500,000) and a flow rate of 0.6 ml/ml. The detector was UV based at 280 nm with an attenuator absorbance range of 0.32. The voltage was 2 mV at a speed of 1 cm/min, the eluent 0.2m Na2SO4 at a pH of 7.25, and the amount of inject volume used was 20 l. The sample loop was cleaned before sample injection by adding 85 l mobile phase avoiding any air bubbles. Only 37 l of sample had to be injected at the time by pressing the plunger on to the 5 l mark on the syringe to make sure that no bubbles were created. In order to proceed with the HPLC operation, a 2 mg/ml solution was prepared for the analysis and the sample was injected into the loop and allowed to operate. Chromatograms of few standards were prepared to create standard for the sample injected into HPLC. This standard set up a linear relationship between molecular weight and elution volume via comparison of the logarithmic value of molecular weight and the ratio of (Ve/Vo). Crystal Formation Another of the techniques used to obtain the crystal structure of lysozyme was the Hanging Drop method. Lysozyme was used as the crystallization choice in this procedure. The crystallization conditions that were tested contained two kinds of salts, NaCl and NaI, at different pH 5,7 and 9. A 24 well plate was assembled to test the decreasing concentrations of the salts at a single pH. Each of these pH values were tested with six different concentrations of both salts. Solutions of 2, 4, 6, 8, 10 and 24% salt concentrations were prepared to tests the crystal formation abilities of both NaI and NaCl. The pH prepared for the group was NaCl at a pH of 9. The wells were allowed to settle for an extended period of time while crystal developed.

Results: Physical Characteristics The following experiment comprised a series of techniques in order to purify to a great extent aldolase. The High Performance Liquid Chromatography was first used to characterize aldolase and test for molecular weight. This analysis was used to collect some data for void volume (Vo), molecular weight of each standard peak, and to calculate the elution volume (Ve) of each peak as well. Before testing, the sample prepared was rated at 2 mg/ml, which after calculating the 37 l placed into the HPLC during analysis, came to 0.074 mg (74g) of aldolase sample. A few standards were added previously to the HPLC in order to obtain a MW standard chromatogram. The flow rate set for this column was 0.6 ml/min at 280 nm, the voltage was 2mV at a speed of 1 cm/min, and injected volume used was 37 l. The equation C1V1=C2V2 was used to calculate the initial volume to find the ratio of the dilution for the peak. In the HPLC operation, the final concentration had to be 2 mg/ml for solution of the sample injected and the final volume was 200 l with 0.2M Na2SO4. The initial concentration of the sample peak was found to be 6.8759 g from previous experiments. The initial volume found for the peak in this HPLC operation turned out to be 0.058 ml. The final concentration given for the solution of the sample had to be subtracted to the final volume found. The ratio of the dilution in this case was 142/58. The Vo value was obtained by using distance of 10 cm and multiplying that by the flow rate of 0.6 ml/min.

Figure 1: The molecular weight for all the visible peaks in the sample run found using the void volume and the elution volume. The calculation of the elution volume over the void volume gave as a result the molecular weight of each peak. In this case, the Blue Dextran was first calculated using the standard
molecular weight. The results obtained for the void volume from Blue Dextran was used to calculate the elute volume of the other standards. It possessed the highest molecular weight and peak.

Standard Peaks

Standard Molecular weight (Da) 12,400 66,000 200,000 2,000,000

Void Volume (Vo) (ml) 6.18 6.18 6.18 6.18

Elution Volume (Ve) (ml) 11.94 9.84 8.22 3.90

Cytochrome C Albumin, Bovine Serum B-Amylase, Sweet Potato Blue Dextran

Figure 2: The logarithm of molecular weight of each standard and the calculated (Ve/Vo) results. This table contains the information used to construct a standard plot with log(MW) against the (Ve/Vo). The equation (Ve/Vo) is used to find molecular weight as well. Standard Peaks Standard Molecular weight (Da) 12,400 66,000 200,000 2,000,000 Log (MW) (Ve/Vo)

Cytochrome C Albumin, Bovine Serum B-Amylase, Sweet Potato Blue Dextran

4.09 4.82 5.30 6.30

1.93 1.59 1.33 0.63

Graph 1: Standard Plot for logarithm of molecular weight against the (Ve/Vo) equation. A standard plot of the logarithm of molecular weight of each standard against elution and void volumes had to be constructed. This standard plot was useful to identify the molecular weight of each peak.

Molecular Weight Standards for HPLC

7 6 Log(Molecular Weight) 5 4 3 2 1 0 0 0.5 1 1.5 Elution Volume (Ve/Vo) 2 2.5 y = -1.6716x + 7.4176

The distance found from the start point in the graph to the peak of aldolase turned out to be 14.9 cm. The distance from the second peak in the graph to the start multiplied with the flow rate of 0.6 ml/min was 13.5 ml, which represented in this case the elution volume. The retention time for the components represented the time elapsed from the time of injection to the detection. The retention time was calculated by dividing the distance from point of injection to peak height over the chart speed. The chart speed was 1 cm/min and the distance on the start point to peak of aldolase 14.9 cm. This result was very important in order to calculate the elution volume. The area under the tetrameric aldolase peak was found to be 20.625 cm2. The speed at which sedimentation velocity is run depends on the molecular weight and shape of the molecules. By determining the slopes of the graphs for native and acid-treated aldolase, the molecular weight of each can be determined. The slope of the acid treated aldolase came to 0.0006 while the native aldolase came to 0.0002. The sedimentation velocity was determined using these slopes. The sedimentation velocity for the acid-treated aldolase turned out to be 9.68 x 10^-8 m/s, and the native aldolase turned out to be 2.9 x 10^-7 m/s.

Figure 3: The molecular weight value of the native and denatured aldolase for each of the three methods used: HPLC, SDS-PAGE and Sedimentation velocity were shown in the table below.

Method HPLC SDS-PAGE Sedimentation velocity (Native) Sedimentation Velocity (AcidTreated)

Molecular Weight 147,293 45,640 158,609 62,125

Figure 4 : Determination of Sedimentation Velocity Type of Aldolase Native Aldolase Acid-treated Aldolase Sedimentation Velocity (m/s) 2.9 x 10^-7 9.68 x 10^-8 Slope of log (x) vs. Time 0.0002 0.0006

Figure 5: Information used for sedimentation velocity of acid-treated aldolase Time (min) 8 24 48 80 112 a 5.6 6.2 6.7 7.6 8.4 x 6.26 6.32 6.37 6.46 6.54 Log (x) 0.796 0.8007 0.804 0.810 0.8156

Figure 6: Information used for sedimentation velocity of native aldolase Time (min) 8 16 32 48 a 6.3 7.3 8.9 10.1 x 6.33 6.43 6.59 6.71 Log(x) 0.801 0.808 0.819 0.827

In this tables, the value of a represented the distance from the start point to the first peak and the x value was calculated using the results from the a value and the 10X magnification

Graph 2 : Native Aldolase Sedimentation Velocity Standard Plot

Plot for Log(x) against the Time for Native Aldolase

0.83 0.825 0.82 0.815 0.81 0.805 0.8 0.795 0 10 20 30 Time (tm) 40 50 60

Log(x)

y = 0.0006x + 0.7971

Graph 3: Acid-Treated Aldolase Sedimentation Velocity Standard Plot

Plot for Log(x) against the time for Denatured Aldolase

0.82 0.815 Log(x) 0.81 0.805 0.8 0.795 0 20 40 60 Time (tm) 80 100 120 y = 0.0002x + 0.7953

In the Lyzsozyme crystal formation process, NaCl and NaI were the two crystals that were used at different pH values to obtain an optimal crystallization. For NaCl, no crystals were formed at a pH of 5 and 7 under any concentration. A different scenario was observed in pH 9, where crystallization was obtained for this salt. The formation of crystals was observed in every concentration, and the predominant shape was monocyclic instead of the tetragonal shape it was supposed to have. By comparing these results with a paper, both concentrations 4 and 6% NaCl at a pH of 9 formed clusters of pure crystals. Any of these sets could be used as a method of crystal analysis. For NaI, the 2 and 4% concentration of the salt did not show any crystal formations while the other rest did show crystals. These crystals showed a pure tetragonal and diamond shape. At pH 7, the NaI salts had crystal formation for all concentration, and they did not show a define shape (disperse crystallization throughout the drop). On the other hand, the crystals formed at pH 9 showed a tetragonal shape. For NaI, pH 5 seemed optimal for growth. For NaCl, the appropriate conditions were at pH 9 at all concentrations. The other salt NaI showed appropriated conditions at pH 9 any concentration and pH 5 at 10%. These two subsets were the only criteria for the formation of appropriate crystals needed to understand the crystal structure of Lysozyme in NaCl and NaI. The last major steps taken to characterize aldolase were isoelectric focusing and C terminal analysis. For this IEF, the pI at 24C value of aldolase was obtained by a group of standards that were run with the sample. The pI of the sample was assessed at 8.15. In this case, the pI obtained was a little bit high. Graph 4: The following graph contains the values for the distance (mm) from the positive electrode and the pI.

Plot for pI and the distance from Positive Electrode

10 9 8 7 6 5 4 3 2 1 0 0 20 40 60 Distance (mm) 80 100 120

pI 3.5 4.55 5.2 5.85 6.55 6.85 7.35 8.15 8.45 8.65 9.3

pI

Distance (mm) 96 80 73 69 57 41 31 19 14 10 5

For C-terminal analysis, the C-terminal amino acid was removed by exposing it to carboxypeptidase and tested by a 2 way TLC assessment. The solvent point for this C-terminal residue identification was 11 cm. The spots were added to the plate 3 cm away from the start point. Four sample tubes containing the assay II components were mixed with carboxypeptidase and some aldolase solution. After waiting 0, 2, 5, and 15 minutes, 10 l of Assay II and 500 l to Dowex were

removed from these sample tubes. A PD value was also measured from a solution lacking of carboxypeptidase to be used as a control.

Figure 7: The absorbance measurement for all these samples was measured at each time.
Time (min) PD 0 2 5 15 Absorbance 0.389 0.408 0.241 0.158 0.069

Figure 8 : In this silica TLC plate, the sample spots turned into a reddish color. Comparing the Rf values was also another way to assess the C-terminal end. The Rf value found after 15 minutes for the sample was similar to the Rf value of standard tyrosine. The results showed that the C-terminal amino acid was tyrosine. The Rf value for that sample at 15 minutes was 0.445, which is very close to a pure sample of tyrosine. Samples Alanine Glutamic acid Glycine Methionine Proline Threonine Tyrosine PD 0 minutes 2 minutes 5 minutes 15 minutes Distance (cm) 2.7 2.3 2.1 1.4 2.1 2.4 4.9 2.1 3.8 4.6 4.8 4.9 Rf value 0.245 0.209 0.191 0.127 0.191 0.218 0.445 0.191 0.345 0.418 0.436 0.445

The carboxypeptidase was necessary to release the C-terminal amino acid from aldolase molecule. Before adding this carboxypeptidase, the amino acid was still attached to this molecule, but after treatment the amino acid was able to bind to the Dowex Column that it was exposed to. By using this method, all aldolase would be washed off while the remaining tyrosine would remain present until the conditions are met for it to be collected.

Figure 9 : Isoelectric Focusing Gel

Figure 10: SDS PAGE Gel

Figure 11 : Silica TLC Plate

Discussion: Physical characteristics A SDS-PAGE separates proteins based on their primary structure or size, but not the amino acid sequence. The SDS or sodium dodecyl sulfateis a detergent that can dissolve hydrophobic molecules and has a negative charge attached to it. Through the exposure to the charge, the proteins will diffuse through the gel and form the bands depending on their molecular weight. There some bands that are typical of proteins with lots of hydrophobic regions or non-protein substituents, and other bands are typical of a soluble protein. The diffuse or dissaperaance of the bands mainly depends on the acrylamide not polymerized, the wells are not rinsed out, and if the samples are destroyed. During the process of SDS-PAGE on various fractions tested, it was easy to observe the purification of the protein over different stages of isolation. There are some bands that might exhibit the characteristics of an impure substance since the gel had the presence of various bands and a gradient that seemed to exist through it. As the protein was more and more purified, less bands seemed to be present in the gel. There were two methods, ion-exchange chromatography and ammonium sulfate precipitation, that were done for a better purification of the protein and an improvement of the protein specific activity. When the run was already completed, the stain that was chosen was standard Coomassie Brilliant Blue R-250 because it is an easy dye to see with naked eye and tends to dry quickly. The sample after exposure to the dye became unusuable since the dye stains directly the protein. This made the sample in the gel usable for only the gels purpose. There were different band intensities observed on the different fractions. The intensity of bands for fraction I, II IV, V, VIA, VIB, and water showed very faint bands and seemed to have some impurities in it since not an specific bold band can be seen. The fraction III showed a very strange band formation since protein bands looked bigger and longer traveled invading the other fractions space. A possible factor for this outcome could be at the time of the addition of the fraction sample to the well, it might have probably gotten a greater amount of the sample or some of the other fraction got into it. The peak fraction was the only of the fractions that actually showed one band meaning that it was well purified. After receiving the results for the HPLC procedure, other two peaks were observed other than the one expected for the aldolase sample. The possible explanation was due to other samples present in the aldolase, degradation products present, and the presence of other proteins in the sample that might mimic aldolase function in the purification process. In order to improve the column and increase the specificity and resolution, there are some possible things that can be done on the column. By having a longer column length, a smaller resin bead size, a slower flow rate, and a smaller fraction size the resolution of the compounds on the column can improve in a great way. By using these criteria, any other protein present in the column could be separated from the aldolase. The calculation of the molecular

weight of aldolase under the three different methods (HPLC, SDS-PAGE, and Sedimentation velocity) showed that the best method to obtain the MW values was HPLC. The other methods were also good to calculate the MW of the protein, but HPLC was the best. A native aldolase was considered a combination of aldolase molecules and a pure form of the protein. On the other hand, the acid-treated aldolase represented a disintegration of the structure of the protein. In order to obtain the crystallization of Lysozyme, two salts were used: NaCl and NaI at different pH calues of 5, 7, and 9 under diverse concetrations 2, 4, 6, 8, 10, and 12%. These were some conditions that essential for the optimal growth of crystals. For NaI, pH 5 seemed optimal for growth and showed appropriated conditions at pH 9 any concentration and pH 5 at 10%. The appropriate conditions for NaCl were at pH 9 at all concentrations. The appropriate conditions were found to reflect the concentrations that have most favorable conditions for a proper crystallization. Chemical Characteristics Based on the purity of the sample found in the IEF gel, it seemed like there was not pure aldolase present yet. Due to the amount of bands observed, the sample most likely was impure due to the gradient that was created during the run. These other bands can be observed due to the degradation of aldolase present or most probably by an impurity present in the sample at the time of the run. The results from the IEF gel demonstrated that the sample at a pH of 7.4 had a positive charge. At this pH, the sample would tend to go to the anode where there is a positive charge but since this is a positive charge sample, it instead move more to the cathode side. In the C-terminal analysis, the terminal residue was found to be tyrosine. The results in the TLC plate showed a spot for the tyrosine amino acid, which catalogued it as the amino acid of identification. During a period of 5 minutes, we were able to observe that the majority of aldolase successfully excised the tyrosine and after 15 minutes the aldolase was almost a hundred percent excised. By comparing these results to one of the references, the solution after 15 minutes was the most effective to show carboxypeptidase activity since it had a near complete reduction in aldolase activity from kinetic analysis. Also, it was effective to show a time point analysis for C-terminus amino acid determination.

References: 1. Lorentzen, Esben. Mechanism of theStructural Analysis of Reaction Intermediates. ACS Publications. (2005). Wed 30 Nov. 2010 2. Lubini, Ditmar. Paracatalytic modification of Aldolase. (1979). PNAS. Wed. 30 Nov. 2010 3. Pugh, E.L. et. Al. (1967). Function of tyrosine residues in rabbit muscle Aldolase. Albert Einstein College of Medicine, Bronx, New York. Wed 30 Nov. 2010 4. Richard, O.C. et. Al. (1961). Journal of Biological Chemistry. 236, 3185-3191. Takaheshi, Kenji. The Reaction of Phenylgloxal with Arginine Residues in Proteins. (1968). The Journal of Biological Chemistry. Wed 30 Nov. 2010