The Physical and Chemical Properties of Aldolase Isaac Kim Partner: Shenhao Chen April 22, 2016 Due and Submitted: April 22, 2016

Transcription

1 The Physical and Chemical Properties of Aldolase Isaac Kim Partner: Shenhao Chen April 22, 2016 Due and Submitted: April 22, 2016

2 Abstract To follow and predict the functions of proteins, the physical and chemical properties of these proteins must be understood. In this experiment, the physical properties of purified rabbit muscle aldolase were probed by gel electrophoresis, high performance liquid chromatography, and sedimentation velocity. From gel electrophoresis, the molecular weight of the subunit of aldolase was found to be 43,805. High performance liquid chromatography found the tetramer to be 156,159 Da, while sedimentation velocity found aldolase to be 153,473 for the tetramer and 44,598 for acid treated aldolase (expected monomer). The chemical properties were found using isoelectric focusing and C-terminal residue identification. By isoelectric focusing, the isoelectric point of aldolase was found to be C-terminal residue identification by carboxypeptidase A found that the C-terminal residue of aldolase is tyrosine. Comparing these values to respective values found previously in other experiments, it was confirmed this experiment was a success in characterizing rabbit muscle aldolase. Introduction Aldolase is an enzyme that catalyzes the reaction of fructose-1,6-bisphosphate to dihydroxyacetone (DHAP) and glyceraldehyde-3-phosphate (Ga3P). 4 The protein is composed of 4 monomers of 39,111 Da weight and comes together to form a tetramer at 156,844 Da. At the ends of this 363 amino acid long protein are proline at the N-terminus and tyrosine at the C- terminus. The proteins isoelectric point is known to be around In this experiment, aldolase that was previously purified from rabbit muscle was analyzed. The aldolase was first extracted from the rabbit muscle by KOH and EDTA. The resulting solution was then purified by salt fractionation with 50-60% ammonium sulfate and then dialyzed to remove salt and small impurities. This was then purified farther with affinity column

3 chromatography using phosphocellulose resin. During each step of purification, fractions were taken and examined by activity assays to monitor the purification process. With this, the goal of this experiment was to explore the different characterization techniques available. With high performance liquid chromatography (HPLC), sedimentation velocity centrifugation, and SDS-polyacrylamide gel electrophoresis (PAGE) the physical characteristics of aldolase like size and subunit associations can be explored. With C-terminal analysis and isoelectric focusing, the terminal amino acid and side chains of the amino acids within the proteins can be examined. Materials and Methods SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Polyacrylamide gel electrophoresis (PAGE) was done by combining a 12% acrylamide separating gel with a 4% acrylamide stacking gel. The 12% acrylamide separating gel was created by combining 3.35 ml distilled water, 2.5 ml Tris-HCl (1.5 M, 8.8 ph), 100 ul SDS (10% w/v), 4 ml Acrylamide (30%), 53 ul Ammonium persulfate (10%), and 6 ul of TEMED. The 4% stacking gel was created by combining 6.1 ml distilled water, Tris-HCl (0.5 M, 6.8 ph), 100 ul SDS (10% w/v), 1.3 ml Acrylamide (30%), 53 ul Ammonium persulfate (10%), and 12 ul of TEMED. The samples run on the gel were fractions obtained from the purification of rabbit muscle aldolase experiment. Fractions I (14.26 mg/ml), II (3.205 mg/ml), III (3.44 mg/ml), IV (39.14 mg/ml), V (29.33 mg/ml), wash (1.45 mg/ml), 6A (3.87 mg/ml), and 6B (2.28 mg/ml) were appropriately diluted to 0.5 mg/ml with distilled water. Appropriate amounts of these fractions were then treated with 2x SDS sample buffer (5 ml Tris-HCl [0.5M, 6.8 ph], 2.2 ml glycerol, 3.2 ml SDS [10% w/v], 0.8 ml 2-β mercaptoethanol, and 0.2 ml 1% Bromophenol blue) and heated at 95 C for 4 minutes. A molecular weight standard (12k 225k Da) and 20 ul of each fraction

4 were added to the gel. Using sample calculation 4, about 4 ug of protein were added to the gel for fractions I-V and 2 ug of protein were added from the gel for fractions 6A, 6B, and wash. The gel was run at 200 volts for approximately 45 minutes. The gel was then stained using Standard Coomassie Brilliant Blue R-250 (0.1% Coomassie Blue R-250 in 40% MeOH, 10% HOAc) for 30 minutes. This was then rinsed with distilled water and then destained with 40% MeOH, 10%HOAc overnight. High Performance Liquid Chromatography (HPLC). Fraction 6A (3.87 mg/ml) from the aldolase purification experiment was diluted down to 2 mg/ml using 0.2 M Na2SO4. About 20 ul of this diluted solution was analyzed by HPLC using the Lab Alliance HPLC unit. The instrument contained a Progel-TSK G3400SW column which separated materials of molecular weights 10, ,000. The detection unit within the instrument used UV light at a wavelength of 280 nm and an attenuator of absorbance range of The recorder used had a voltage of 10 mv and a chart speed of 1 cm/min. The eluent used was Na2SO4 (0.2 M, 7.25 ph) at a flow rate of 0.6 ml/min. Crystal Formation. The protein lysozyme was crystallized using the hanging the drop method. Sodium chloride (NaCl) or sodium iodide (NaI) were used at different salt concentrations (2, 4, 6, 8 10 and 12%) at 3 different phs of 5, 7, or 9 to grow the lysozyme crystals. A salt solution, either NaCl or NaI, was diluted down from 24% using deionized water to an appropriate salt concentration (2, 4, 6, 8 10 and 12%) so that the final volume was 500 ul. This was then combined with 500 ul of NaH2PO4 (0.1 M, ph of 5, 7, or 9). On a SigmaCoted (silicon/heptane) cover slip, 5 ul of lysozyme (25-30 mg/ml) was added to the center. An equal volume of salt-buffer solution was added to the drop of protein. This was then quickly flipped onto a greased well containing the respective salt-buffer solution. This was repeated until all the

5 different mixtures of salt concentrations and phs were covered. This was then allowed to sit for 1 week at a stable temperature. Results HPLC. To estimate the molecular weight of the purified aldolase in fraction 6A, gel filtration was done by HPLC. Fraction 6A (3.87 mg/ml) was diluted down to 2 mg/ml and 30 ul of this was injected into the HPLC instrument. Using sample calculation 4, this was a total of 60 ug of protein injected. After allowing the sample to run through the column, one peak was seen 13.1 cm from point of injection. Through triangulation, this peak was found to have an area of about 4.78 cm 2. The elution volume (Ve) for fraction 6A was calculated using sample calculation 2 and was found to be 7.86 ml. This was then compared to molecular weight standards (blue dextran, Bovine Serum Albumin (BSA), Cytochrome C, and Β-amylase) that were previously run on the HPLC. The void volume (Vo) was found to be 5.88 ml by using sample calculation 1. The data obtained from blue dextran was used to calculate Vo since it will not pass through the column due to its size. The data from the standards can be found in Table 1. Using this, a standard curve was created and is found in Figure 1. Table 1: Molecular Weight Standards for HPLC Protein Standard Molecular Peak Distance Elution Volume Ve / Vo weight (Da) (cm) (Ve) [ml] Cytochrome C 12, BSA 66, Β-amylase 200, Blue Dextran 2,000,

6 Log (Molecular Weight) HPLC Molecular Weight Standards y = x Ve/Vo Figure 1: HPLC Molecular Weight Standards. The standards of Cyt. C, BSA, B-amylase, and Blue Dextran were passed through a HPLC column. The resulting data was used to create a standard curve using sample calculations 1-3. The ratio of Ve/Vo for the aldolase sample was calculated to be Using this and the standard curve, the molecular weight of aldolase found in fraction 6A was calculated to be 156,159 Da. Standard aldolase was also run on the HPLC. About 20ul of 20 mg/ml protein standard was injected into the HPLC. Using sample calculation 4, this was about 40 ug of protein. This yielded a peak with an area of 4.13 cm 2. Using sample calculation 9, it was found that about ug of the 60 ug of aldolase injected was a tetramer. Comparing this ratio to fraction 6A, roughly 2.99 mg/ml of the 3.87 mg/ml protein concentration is a tetramer. SDS-PAGE. Fractions I V, 6A, 6B, and wash from the aldolase purification experiment were applied to an electrophoresis gel after being appropriately diluted, heated, and treated with SDS. After running the gel and staining with Coomassie Brilliant Blue, the gel in Figure 2 was obtained.

7 Log ( Molecular Weight) W M 6A 6B Figure 2: SDS-PAGE of aldolase purification fractions. Fractions from the aldolase purification were diluted and denatured with SDS and applied to a gel. This was then stained with Standard Coomassie Brilliant Blue and visualized. From left to right Fraction I, II, III, IV, V, wash (W), molecular weight standard (M), 6A and 6B. Comparing the lanes of the gel, the lanes containing 6A and 6B had the least amount of bands present. These lanes also contained the most intense bands, hinting to the highest concentration of a single protein. From this, the bands present within fraction 6A will be used for aldolase molecular weight determination. Using the molecular weight markers run on the gel, the following molecular weight curve, Figure 3, was generated. Gel Standard Molecular Weight Markers y = x Distance Traveled Figure 3: Molecular weight standard graph. With the fractions, a molecular weight standard was added to the SDS-PAGE. The molecular weight standards ranged from 12 kda 225kDa. The 102, 150, and 225 kda markers were omitted due to the bands over lapping at the top of the gel.

8 Log (Peak Distance) The distance traveled by the bands within the fraction 6A lane were seen to 3.9 and 2.2 cm. Using Figure 3, the molecular weight of the two bands were calculated to be 43,805 Da and 61,144 Da respectively. Sedimentation velocity. The third method used to determine the weight of aldolase was sedimentation velocity by an ultracentrifuge. Aldolase and acid treated aldolase were both run at a speed of 60,000 rpm in order to calculate the molecular weight of each species. Figure 4 shows the graph of time plotted against the log (distance traveled by peak). Sedimentation Vecolity of Aldolase and Acid treated Aldolase y = x y = x Time (min) Linear (Native Aldolase) Linear (Acid Treated Aldolase) Figure 4: Sedimentation velocity of aldolase and acid treated aldolase. Aldolase and acid treated aldolase were run in an ultracentrifuge at 60,000 rpm. At appropriate time points, measurements were made during each run. The distance of the peak from inner reference line was recorded and is reported as log (peak distance) vs. Time (min). From Figure 4, it was seen that d(log peak distance) dt m of aldolase was and for acid treated aldolase. Using sample calculation 5, 6, and 7 the sedimentation coefficient (SObs), the normalized sedimentation coefficient 20 C in water (S20,w), and the molecular weight can be calculated.

9 Table 2: Results of velocity sedimentation of Aldolase and Acid Treated Aldolase Protein d(log peak distance) SObs (s) S20,w (s) Molecular dt m Weight (Da) Aldolase * * ,473 Acid Treated Aldolase * * ,598 From the data obtained from the velocity centrifugation, the aldolase was found to be 153,473 Da while the acid treated aldolase was found to be 44,598 Da. Table 3: Summary of molecular weight findings Characterization Technique Native Aldolase (Da) Denatured Aldolase (Da) HPLC 156,159 - SDS-PAGE - 43,805 Sedimentation velocity 153,473 44,598 Table 3: High Performance Liquid Chromatography (HPLC) was used to determine the weight of native aldolase. No denatured proteins were run in this. SDS-PAGE denatured the protein to its subunits in order to find the weight of the subunit. No native proteins were available for this due to the addition of SDS. Sedimentation velocity of native and denatured aldolase was done to determine the weights of both tetramer and monomer respectively. Isoelectric Focusing (IEF). In order to identify the isoelectric point of aldolase, isoelectric focusing was done. Fraction 6A (3.87 mg/ml) was diluted to 2 mg/ml and 4 ul was applied to the gel. The resulting Gel can be found within Figure 5.

10 pi S M 6A Figure 5: Isoelectric focusing gel. Fraction 6A (3.87 mg/ml) was diluted to 2 mg/ml and 4ul of this was applied to the gel.. Sigma aldolase (S) and a pi markers (M) were also added to the gel and are appropriately labeled above. After running the gel, the gel was visualized by staining with Coomassie Brilliant Blue Using the pi markers that were run in unison with Sigma Aldolase and Fraction 6A, the following standard found in figure 6 was generated. pi Markers on Isoelectric Focusing Gel y = x Distance Traveled (mm) Figure 6: pi Markers on Isoelectric Focusing Gel. On the gel, pi markers were added along with the Fraction 6A and Sigma Aldolase. The pi markers used were Amyloglycosidase (pi 3.5), Trypsin inhibitor (4.55), B-Lactoglobulin A (5.2), Bovine Cabonic anhydrase B (5.85) human carbonic anhydrase B (6.55), myoglobin acidic band (6.85), myoglobin basic band (7.35), lectin-acidic band (8.15), lectin-middle band (8.45), lectin-basic band (8.65), and trypsinogen (9.3) were used. The first three markers were omitted in the standard curve due to their poor visibility. Measurements were made from the positive electrode.

11 Sigma aldolase and Fraction 6A (most intense band) both had one strong band present 61 mm from the positive electrode. Using the standard curve found in Figure 6, it was found the pi for Sigma Aldolase and the aldolase present in Fraction 6A is C-terminal Residue Identification. The next chemical property of aldolase that was observed was the C-terminal residue. In order to find this, carboxypeptidase A was employed to digest a commercial aldolase (8mg, 3.0mL). During the digestion of aldolase, the reaction was monitored at time points 0, 2, 5, and 15 mins along with a predigest sample. After the reaction was completed, this was then run on a silica TLC plate along with amino acid markers. The TLC plate can be seen in Figure 7. Figure 7: TLC plate of aldolase digestion with carboxypeptidase A. Commercial aldolase (8mg/3ml) was dialyzed against ammonium bicarbonate buffer (0.2M, 7.8 ph) overnight. This was then digested with carboxypeptidase A (100 U/ml); the reaction was monitored prior to digest and at time points 0, 2, 5, 15 mins. The reaction was stopped using Dowex 50 ( mesh H+ form). This was then run on a TLC plate along with amino acid markers. The markers are alanine, glutamate, glycine, methionine, proline, threonine, and tyrosine from lanes 1-7. Lane 8 contains the predigest while 9-12 are time points between 0-15 mins.

12 Examining the TLC plate, it was seen that as the longer the carboxypeptidase was allowed to react with the aldolase, the more amino acids were digested. This was evident by the intensities and the number of the spots on the TLC plate at later time points. Comparing the predigest aldolase lane with the lanes containing the different time points of the reaction, a spot that correlates with tyrosine, seen in lane 7, is visualized. Using sample calculation 8, the Rf value for the first appearing spot is 0.483, which matches the Rf value for the tyrosine standard spot. This spot is the first to appear as the reaction progressed and grew more intense as the reaction went on. Therefore, it is concluded that the C-terminal residue of aldolase is tyrosine. Crystal Formation. To find the optimal conditions for growing lysozyme crystals, different salts, salt concentrations, and phs were used. For NaCl at a ph of 5, it was observed that crystals formed at all different salt concentrations. These crystals had a tetragonal shape, were clear, and increased in size as the salt concentration increased. When the ph was increased to 7, one clear tetragonal crystal was seen at 4% NaCl salt concentration while other concentrations did not produce any crystals. At a ph of 9, a combination of clear tetragonal and hexagonal crystals were seen across the NaCl concentrations. One exception was seen at 10% NaCl, where an irregular shape was seen. The size remained relatively constant across the concentrations. For NaI at a ph of 5, irregular shaped crystals were seen across all concentrations of NaI. These crystals were yellow and progressively got smaller as the concentration of NaI increased. At a ph of 7, the 2% NaI concentration yielded small clear irregular crystals. As the concentration increased, the crystals became larger and more shard like. The crystals remained clear up until a concentration of 6%, but at concentrations 8-12%, the crystals became yellow. At a ph of 9, the crystals that were grown were clear and a mix of orthorhombic and hexagonal shapes. These stayed relatively similar across the salt concentrations.

13 Sample Calculations 1. Void Volume (Vo) = Retention Time (mins) x Flow Rate (ml/min) a. Flow Rate = 0.6 ml/min b. RT = [Distance from point of injection to peak height] Chart Speed ( cm min ) i. Chart Speed = 1 cm / min 2. Elution Volume (Ve) = Retention Time (mins) x Flow Rate (ml/min) a. Flow Rate = 0.6 ml/min b. RT = [Distance from point of injection to peak height] Chart Speed ( cm min ) i. Chart Speed = 1 cm / min 3. Ve Vo = Elution volume / Void volume 4. Concentration ( mg Protein Content (mg) ) = ml Volume (ml) 5. SObs =( d(log peak distance) ω 2 )* ( ) dt m a. Where ω = rpm * 2π, rpm = 60, S20,w= SObs ( ƞt ƞ 20,w ) ( ƞ ƞt ) 1 ρ 20,w 1 ρ tw a. ƞt ƞ 20,w = 1.02, ƞ ƞt = , = cm3 /g, ρ 20,w = g ml, ρ tw = g ml 7. Molecular Weight (MW) = R T S 20,w D(1 ρ) a. D = 4.67 * 10-7 (cm 2 /sec), ρ = g ml R= * 10 7 ergs / (mol * K), = cm3 g 8. Rf = (migration of sample) / (migration of solvent front), T = 20 C (293 K),

14 9. (Amount of injected standard) / (area of standard peak) = (Amount new peak/ area of new peak) Discussion A powerful tool used to analyze proteins and protein mixtures is gel electrophoresis. This method provides information about the size of DNA (or proteins) and is able to display the different components within a solution. Due to the equal charge per length and the long rod like structure of DNA, it will move through the gel due to the applied voltage and separate based on the sieving of the gel. Thus by controlling the pore size within the gel, the separation of components can also be controlled. A larger concentration of acrylamide in a gel will result in smaller pores making the range of separation smaller, while a smaller concentration will result in larger pores and a larger molecular weight range of separation. While DNA are long rods with equal charge per length by nature, proteins must be treated to have these traits. By using sodium dodecyl sulfate (SDS), hydrophobic regions of proteins are unwound due to the amphipathic nature of SDS. A negative charge is also distributed evenly across the protein by SDS. By using β mercaptoethanol, disulfide bonds are reduced and broken. Also applying additional heat will break any remaining intramolecular bonds. From this, a protein can now be separated by the gel sieve instead of the length of denatured protein. 3, 6 After running the gel, it must be stained in order to visualize the bands within the gel. The staining technique chosen in this experiment was a positive stain (where the bands themselves are stained) called Coommasie Brilliant Blue. Although different techniques like copper and zinc stains (negative stains that stain the gel) exist, these methods are often hard to visualize. The caveat to this method the ability to retrieve proteins after staining. However, in our experiment the protein was no longer required after visualization, thus the positive stain was chosen.

15 Comparing the bands within the gel, it was seen that the most intense bands occurred within the lanes containing fractions 6A and 6B. These lanes also contained the least amount of bands. Since each fraction was collected over the process of isolating and purifying aldolase from rabbit muscle, the data seen on the gel proves that the purification process was indeed successful. The decrease in bands from fraction I to 6A shows the elimination of impurities, while the brightness of the bands in 6A and 6B show that the aldolase is concentrated within these fractions. One important part to note is the second (less intense) band seen within these lanes. The molecular weight determined for this band was about 61,144, and could represent an aldolase tetramer that did not fully dissociate or impurities that co-eluted in the purification process. This could be improved by increasing the SDS concentration, allowing the SDS to react with protein longer, or adding a step to purification. However, the band s intensity was only a fraction of the aldolase band, and therefore is not a significant portion of the fractions 6A and 6B. After the sample was run on the HPLC, one observable peak was seen on the graph. However, if a second peak was observable within our data, it most likely represents an impurity or the tetramer breaking into its subunits. During the purification of aldolase from rabbit muscle cells, some proteins with similar properties as aldolase, like similar affinity for a substrate and salt tolerance, may have co-eluted and remained within the solution. Overall, the resolution of our results were good and no overlap of peaks occurred since only one observable peak was seen. If however the resolution was poor, an option to enhance the resolution would be to either slow the flow rate of the HPLC or decrease the size of the resin beads. By doing this, it would give the sample more time to interact with the resin and give the resin a larger effective surface area to react with the sample, respectively. Another option would be to use less sample when injecting

16 into the HPLC. By doing this, the sample will be more concentrated and the protein within the sample will be able to access the resin quicker, thus raising the resolution. In Table 3, the molecular weights findings by HPLC, SDS-PAGE, and sedimentation velocity are seen. The known weight for the tetrameric aldolase is 156,844 Da and 39,211 for the monomer subunit. 2 HPLC found that the weight of aldolase was 156,159 Da which is very close to the aldolase tetramer. SDS-PAGE found that the aldolase was about 43,805 Da, which is close to the monomeric subunit of aldolase. Sedimentation velocity found native aldolase to be 153,473 Da and acid treated aldolase to be 44,598 Da, which again are very close to the molecular weights of the tetramer and monomer, respectively. Thus, the 3 methods used provided accurate information of the physical properties of aldolase. However, of the 3 methods, HPLC provided a molecular weight that was closest to an actual value, making it the most reliable technique. The data seen by HPLC, SDS-PAGE, and sedimentation velocity in unison proved that aldolase is indeed a tetramer. Since SDS-PAGE denatures the protein down to its subunits, this will provide the data for the monomer. No denatured species were inserted into the HPLC, thus the data this method provided gave the weight of the entire protein. Since the weight found by the SDS-PAGE is about ¼ of the weight found in HPLC, the protein must be a tetramer. The sedimentation velocity data further proves this. If this protein were a monomer, the SDS-PAGE would have shown a band near 150 kda. Examining the crystallization of lysozyme, it was seen that the best salt for growing crystals is NaCl. Although both salts did yield crystals, NaCl yielded more regularly shaped crystals across the different phs and salt concentrations. NaI did have growth, but the crystals seen in ph 5 and 7 were mostly large shards of irregular shape. Lysozyme crystals grown in NaCl are known to be

17 tetragonal, while NaI produces orthorhombic crystals. 5 When observing the crystals in this experiment, the crystals grown in a ph of 9 were consistent with these results. Thus, the best condition would be NaCl at any concentration at a ph of 9. The 10% NaCl salt concentration did yield an odd result in NaCl at a ph of 9, but this may be attributed to error. If a wrong salt concentration or vibrations were present during crystal growth, this may have cause the erroneous results. Isoelectric focusing found that the pi for aldolase is 8.16, this being very close the actual value of Based on the isoelectric gel, it was determined that the fraction applied was pure. Only one band was seen throughout lane 6, showing a single species was present in our Fraction 6A. However, this single band seen was not a sharp band, but was rather diffuse. Isoelectric focusing allows proteins to move through the gel until they reach their isoelectric points, where the protein has no charge. Thus, anything above the pi of a protein, would make the protein have a negative charge, and anything below would cause the protein to be positively charged. Aldolase is known to have a pi of 8.2, and thus at physiological ph of 7.4, the protein will be positively charged. The isoelectric point of 8.2 is located rather close the well where the sample was injected. Aldolase would have reached the pi point rather quickly and had to sit in this position as the gel continued to run. Due to entropy, this band will diffuse into its surroundings inevitably. However, the diffuse proteins will move back to the pi due to the applied current and charge of protein. The equilibrium between these states most likely cause the diffuse bands seen. Aldolase is known to have tyrosine at the C-terminus from previous studies. 2 From the carboxypeptidase digestion done, it was found that our results also concluded that tyrosine is at the C-terminus. The first amino acid spot seen within the reaction lanes had an Rf value identical to the tyrosine standard spot, showing that this had to be the first amino acid digested on the C-

18 terminal end of the protein. As time went on during the reaction, more peptides were digested from the C-terminal end of aldolase. Comparing the spot intensities of times 0, 2, 5, and 15 mins on the TLC, it was seen that the spots grew darker as the reaction proceeded. This proving the increase of digestion as time went on. When aldolase reacts with FBP to form DHAP and Ga3P, the active site uses the amino acids arginine and lysine during the catalysis. 4 Examining the 363 amino acid sequence of aldolase, it is seen that neither of these two amino acids are close the C-terminal end of the protein. Therefore, the digestion must be allowed to run before it majorly effects the active amino acids. Thus the later time point of 15 min would cause the carboxypeptidase to inhibit the aldolase the most. Acknowledgements I would like to thank Dr. Padala and Melanie Yap for their assistance with my lab work and providing a great learning environment. Thank you for a great semester! References 1. Laemmli, U.K., Nature, 227: (1970). 2. Lai, C., Nakai, N., Chang, D. Science, 183, (1974). 3. Friefelder, D. Physical Biochemistry, W.H. Freeman and Co., San Francisco, Ca Hill, H.A.O., Lobb, R.R., Sharp, S.L. and Stokes, A.M., Biochemical Journal, 153: (1976). 5. Wang, X., Liu, Y., Ching, C. Lysozyme crystallization, ACS, 10: (2010). 6. Weber, K., Pringle, J.R., and Osborn, M., Methods in Enzymology, (1972).