Physical and Chemical Properties of Aldolase
Hye Soo Lee
Partner: Sohee Min
Due date: April 25th, 2014
Date Submitted: April 25th, 2014
ABSTRACT
In order understand the function of protein in the cell, study of physical and chemical
characteristics of a protein are essential. The principal objective of this experiment was to
determine the physical and chemical properties of aldolase. To identify its properties, the
aldolase was purified from rabbit muscle. The molecular weight was determined by 3
different methods: HPLC, SDS-PAGE, and Sedimentation velocity. Two separate
molecular weights were determined by SDS-PAGE and Sedimentation velocity, proving
the aldolase is comprised of multiple subunits. The charge on the protein was determined
to be positive by isoelectric focusing. The molecular weight of the native aldolase was
determined to be 116,103 Da by High Performance Liquid Chromatography and by
Sedimentation Velocity to be 158,781 Da. The molecular weight of the denatured
aldolase was determined to be 42,413 Da by SDS-Polyacrylamide and 62,306 Da by
Sedimentation Velocity. Isoelectric Focusing was used to determine the experimental
value of the isoelectric point of aldolase, which was found to be 8.17. The C terminal was
determined in this experiment to give Tyr. Finally, Lysozyme was crystallized by the
Hanging Drop Method and shown to produce Tetragonal crystals in NaCl and
Orthorhombic crystals in NaI. The experiment was successful in determining the physical
and chemical properties of the enzyme aldolase.
INTRODUCTION
Aldolase is an enzyme that catalyzes the reaction of Fructose-1,6-bisphosphate to
dihydroxyacetone phosphate and glyceraldehydes-3-phosphate. The structure of aldolase
was originally believed as trimer, but recent studies have shown that aldolase is a
tetramer of 4 monomeric, 364 amino acid residues long polypeptide chains. Each subunit
has a molecular weight of 39,211Da for a tetrameric weight of 156,844Da. (4),(5)
The aldolase isolated from rabbit muscle was used in this experiment. The aldolase
enzyme was extracted by hand stirring with cold KOH and EDTA. Salt fractionation by
50 % and 60% ammonium sulfate precipitation was used to purify the aldolase. Later, the
sample was dialyzed using tube (MWCO 14,000 Da), and put into ion exchange
chromatography with phosphocellulose column. The phosphocellulose column has a
bound phosphate group; proteins that bind phosphate interact with this group and move
more slowly through the column while other proteins move quickly and are washed out.
Aldolase was separated from non-phosphate binding proteins in this fashion. Then, Forty
fractions were collected. The fraction with the highest absorbance was identified as the
“peak”. The fractions with highest absorbance were pooled together as Fraction VIA, and
the fractions with low absorbance were also pooled together as Fraction VIB. These steps
provide a pure sample of aldolase to begin identifying its physical and chemical
properties.
The goal of this experiment are to characterize the enzyme aldolase both physically and
chemically. The molecular weights of both native and dissociate aldolse were determined
by use of SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Sedimentation
Velocity, and High Performance Liquid Chromatography (HPLC). Chemical properties
of aldolase were determined by C-terminal analysis and Isoelectric Focusing. Also,
second goal of this experiment was to observe the crystallization of Lysozyme at varying
pH values in different concentrations with two different salts: NaI and NaCl.
Materials and Methods
SDS-Polyacrylamide Gel Electrophoresis (SDS-Page)
An SDS-PAGE gel was made using a 4% stacking gel and a 12% separating gel. The
internal buffer chamber was filled with the running buffer until the gel was completely
submerged while the outer chamber was filled about the half way to the top of the gel.
Each of sample fractions were diluted with distilled water to give a final concentration of
0.5 mg/ml. 24 μl of 2X SDS sample loading buffer was added to tubes labeled with
Fractions I-V and 32 μl of 2X SDS sampled buffer was added to the “wash”, “peak”
fraction, and Fractions VIA and VIB tubes.
Both sets of samples were heated at 95⁰C for 4 min and centrifuged to disrupt ionic and
hydrogen bonds. 20 μl of each samples were loaded into their respective wells along with
a rainbow molecular weight marker.
Then, the gel was run in 1X Running Buffer for 40min at 200V. After the samples were
moved through the gel, the gel was stained using 0.1% Coomassie Brilliant Blue R-250
dye for 30 minutes. Finally, the stain was poured off the gel and rinsed 3 times with
distilled water and it was destained overnight.
High Performance Liquid Chromatography (HPLC)
High performance liquid chromatography using a SSI 210 Guardian HPLC was used with
a Progel-TSK G3000 SW (MW range 10,000-50,000) column.
Before sample injection, the syringe was cleaned multiple times with mobile phase (0.2M
Na2SO4, pH 7.5). The column (Progel-TSK G3000 SW) was also washed 3 times with
85μl of mobile phase to limit contaminating proteins. The sample (33.758 mg/ml) was
diluted to 2mg/ml with mobile phase. Using syringe, 20μl of sample was injected into the
column. Presence of no air bubbles was important.
The detector was set at 280nm with an attenuator absorbance range of 0.32. The eluent
used was 0.2 m Na2SO4 at a pH of 7.25. The flow rate was set to 0.6 ml/min with a 2-mv
recorder set at 1cm/min. As with the SDS-Page, the sample volume being used was
merely 20 µL.
The data was analyzed using a set of standards (β-Amylase(200,000Da), BSA(66,000Da),
Blue Dextran(2,000,000Da), Cytochrome C(12,400Da)). 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
The Hanging Drop method was used for crystal formation “screening”. Lysozyme
was used as the protein sample for the crystallization media. It was tested with two
different salts, sodium chloride (NaCl) and sodium iodide (NaI). The salts were tested at
different pH levels (5, 7, and 9). The various salt concentrations were made using 500 μl
0.1 M NaH2PO4, 24% NaCl/NaI and distilled water. Each of these various salt solutions
(2%, 4%, 6%, 8%, 10%, and 12%) of both salts were tested with different pH values
using 24 well plates.
5 μl of Lysozyme was added to a SigmaCoted cover slip with 5 μl of the salt solution.
Each of cover slips was flipped on to the appropriate well in order to suspend the drops
above the wells. By the Hanging Drop Method, the crystals formed after extended period
of time. Crystal formation was then examined with each different salt and pH using a
light microscope.
Results
Physical Properties
HPLC
The first step to determine the physical characteristics of aldolase, High Pressure Liquid
Chromatography method was utilized to determine the molecular weight of aldolase. The
length to the peak was 14.9cm. The area of the peak was 14 cm2 and corresponded to an
aldolase concentration of 9.21 mg/ml. 2mg/ml of sample was prepared for injection, and
the volume of 20 µL was placed into the HPLC during analysis. This came to 0.4 mg of
Aldolase sample. Since the original protein concentration was 5.1 mg/ml, 0.4mg of
aldolase sample divided by 5.1mg/ml of original aldolase concentration gave 78ul, which
is the total amount of aldolase injected in the sample.
A graph of the normalized elution volume versus the log of molecular weight standards
(Figure 1) was used to determine the molecular weight of the aldolase sample. By HPLC
method, the elution volume (Ve) of the sample was determined by use of the peak that
distance traveled of peak (14.9 cm) with a flow rate (0.6 ml/min). Multiplying these
values gives a Ve value of 8.94 ml. The column volume, Vo, was determined to be 6 ml.
Thus, the Ve/Vo ratio was ended up with 1.49. Using this value, the experimental value
of the molecular weight was calculated to be 116,103.42 Da by the equation of the
standard line. There were no other significant peaks in the HPLC print out, which
indicated monomeric aldolase.
Sedimentation Velocity
The sedimentation values of native aldolase and acid treated aldolase were
calculated and plotted to generate a standard curve. A graph was generated comparing the
log of the peak distance to time of the two types of aldolase (Figure 3) By determining
the slopes of the graphs, the MW of each may be determined. The slope of the graph for
acid treated aldolase was 0.0002 and was used to calculate a molecular weight of 62,306
Da, and the slope of the native aldolase graph was 0.0007 and a molecular weight of
158,781 Da.
SDS Electrophoresis – SDS-PAGE
In order to analyze the subunit molecular weight of proteins that had been denatured and
dissociated by sodium dodecyl sulfate (SDS), polyacrylamide gel electrophoresis was
performed. When SDS binds to the protein, the subunits dissociate into monomers that
are engulfed by negative charges. The proteins are separated only by the molecular
weight since a charge is run through the gel (electrophoresis). The peak fraction was used
in the determination of the molecular weight of Aldolase. Using the equation of the
standard curve generated by the Rainbow Marker, it was found the aldolase had a
monomeric molecular weight of 42,413 Da.
Lysozyme Crystals
In the Lysozyme crystal formation process, various pH values and salt concentrations of
NaCl and NaI were the two crystal formation solvents in order to examine for optimal
crystal structure formation.
For NaCl, at pH 5, no crystal was formed of 2,4, and 6%. Large crystal but unknown
shape was formed in 8%, and very tiny clumps of crystal formed in 10% and 12%. At pH
7, little tetragonal crystals seemed to form under 4%, 6%, and 8% NaCl. At pH 9,
tetragonal crystals were formed at 2% and 4% concentration. For NaI, at pH 5, 2% has
clump of crystal while 4% and 12% have crystal of unclear shape. 6% and 8% have
clump of orthorhombic crystal but not clear as well. 10% has the best structure of
orthorhombic crystal clump. At pH 7, Orthorhombic crystals seemed to form under 4%,
6%, 8%, and 10%. Especially, 8% concentration gave the best crystal formation while
orthorhombic and tetragonal crystals were formed at 4% concentration. At pH 9, all
crystals of different concentrations have the same structure. They all clumped together.
They don’t look like orthorhombic or tetragonal.
Therefore, for NaCl, the appropriate conditions were at pH7 of 4%, 6%, or 10%, and pH
9 of 2 or 4% concentrations. For NaI, the appropriate conditions were at pH 7 at 6%, 8%,
or 10% concentrations.
Chemical Properties
IEF
Protein mixture with different electric charges can be separated by isoelectric focusing
method. The sample was run on an isoelectric focusing gel, and the pI value of Aldolase
was determined by pI markers that were run with the sample. The distances each band
traveled in the Isoelectric Focusing Gel (Image 2) was measured and generate a standard
curve (Figure 4). Our peak appears at approximately 70 mm, as shown in Lane 6.
Plugging this number into the equation generated by the graph, our pI was 8.17.
C-Terminal Identification
The amino acid present at the C-terminal of the polypeptide was identified. A commercial
aldolase solution (8 mg/ 3.0 ml) was hydrolyzed with carboxypeptidase using four
different time points: 0, 2, 5, and 15 minutes. Carboxypeptidase cleaved the C-terminal
amino acid of the polypeptide thereby stopping aldolase activity. This mixture was then
put through a Dowex Resin column to terminate the hydrolysis of aldolase. The Dowex
resin lowers the pH to 2.3 and then binds to the free amino acids. The aldolase activity,
after carboxypeptidase exposure to different time point, rapidly decreased until the 5
minutes, but not changed much after 5 minutes of exposure. (Image 3)
Then, the eluted amino acid products were run on a TLC plate with the standard amino
acids. The result show that the C-terminal amino acid released in the hydrolysis reaction
with carboxypeptidase was Tyrosine. The Rf values and the color of sample were
consistent with the results produced by the tyrosine standard. Since the Rf values of all
the samples were around 0.46, as well as color, are consistent with the tyrosine standard.
The carboxypeptidase exposure was important in releasing the C-terminal amino acid
from the Aldolase molecule. Without the carboxypeptidase, C-terminus would not be
able to analyze since C-terminus remains intact to the molecule.
Sample Calculations
HPLC Data -
β-amylase:
Ve= 13.6 cm x 0.6 ml/cm = 8.16 ml
Vo= 10 cm x 0.6 ml/cm = 6 ml
Ve/Vo = 8.16/6 = 1.36
C-Terminal Analysis –
Rf1 = distance spot traveled/distance solvent traveled
Rf1 sample = 8.3/18 =0.46
Sedimentation Velocity –
Sobs =slope x F
F=0.03838/ω2
ω=(rpmx2π)/60
ω = (60,000 x 2π) / 60 = 6,280 rad/sec
F = 0.03838/6,2802 = 9.732 x 10-10
Sobs Native Aldolase = 0.0007 x 9.732 x 10-10 = 6.812 x 10-13 sec
S20,w = Sobs (ηt/η20w)(η/ηt)(1-v ρ20w/1-v ρtw)
(ηt/η20w) = 1.02
(η/ηt) = 1.1348
(1-v ρ20w/1-v ρtw) = 1.0029
S20w
Native Aldolase = 6.812 x 10-13 x 1.02 x 1.1349 x 1.0029 = 7.908 x 10-13 MW –
(RTs20,w)/D (1 –vp)
MW Native Aldolase = [(8.314 x 107)(293 K)(7.8988 x 10-13)]/[(4.67 x 10-7)(1(0.742x0.998)] = 0.019241/1.212 x10-7 = 158,781 Da
DISCUSSION
Physical Characteristics
During the SDS Electrophoresis, the aldolase is denatured by SDS, and molecular weight
of aldolase subunit was determined. The gel clearly shows that the protein purification
was successful. Fraction I to III, lane 1 to 3, contains much more bands than other
fractions beside “wash”. This shows the presence of lots of protein mixtures (Image 1).
After each fractions, there are less number of bands present in each lane, indicating the
removal of protein mixtures. “Wash” was the collection of proteins that are not capable
of binding to phosphate within phospho-cellulose resin get washed away leaving the
proteins that stay bound to the resin, such as aldolase. Therefore, lots of bands present in
“wash”(lane 6) strongly supports that most of unwanted proteins are washed way. Also,
the band intensities at the approximate location of aldolase is getting darker in each
fraction, and the peak fraction gives the highest intensity while FractionVIA is the second
highest. Presence of only one band with highest intensity at the approximate location of
aldolase in Lane 8 (Peak Fraction), stongly indicates aldolase is the only protein present
and the purification was successful. Lane 9, Fraction VIA, shows only two bands; the top
dark blue band is the aldolase protein and the lower protein is contamination. This shows
that this fraction is pure and the size of the band shows it contains a significant portion of
aldolase. This makes sense since the fractions with highest absorbance were pooled
together and saved as Fraction VIA during the purification process.
Our gel is unique because the bands are making gradient that loaded more in the first few
lanes than the rest. It could be misunderstand as presence of more aldolse in first few
lanes than others. But the truth is, lots of bands indicate lots of unwanted protein.
Once the protein had run through the gal, the stain of choice was standard Coomassie
Brilliant Blue R-250. This method was used because it is a positive stain, which is much
easier to visualize with naked eye than a negative stain. Since it directly stains the
protein, the sample is rendered unusable after exposure to a dye. This doesn’t matter
because the protein did not need to be used further.
From the data in the HPLC procedure, there was no other peak than aldolase peak.
Presence of only one peak indicates that the aldolase is relatively pure. More numbers of
peaks would suggest contamination in the “peak” fraction. The peak had an area of 14
cm2 which corresponded to a total of 78 μg of aldolase injected.
Possible ways to create a high resolution besides getting a new column is lowering the
flow rate. Also, injection of less amount of sample by using more concentrated sample
would help for better resolution. A lower flow rate would give more time for the sample
to interact with the column so that the components could separate better. A reduced
sample size would give less overlap in the sample and hence better resolution.
The molecular weight of aldolase was determined by using three different methods:
HPLC, sedimentation velocity, and SDS-PAGE. From the HPLC methods, molecular
weight of aldolase was determined to be 116103.42 Da. Data from SDS-PAGE showed
the molecular weight to be 42,413 Da. Sedimentation method showed the molecular
weight of acid treated aldolase to be 62,306 Da and the molecular weight of native
aldolase to be 158,781 Da. The differences in molecular weight values in different
methods support that aldolase is a multimeric protein.
Based on the calculations of the MW of Aldolase under different methods, HPLC
provided the most accurate method of showing MW values. (Table 2) While
sedimentation and SDS-Page both provided reasonably accurate values for MW for
calculating values, HPLC was all the more accurate overall. Nonetheless, all of the
values showed that native Aldolase was a combination of Aldolase molecules
interconnected to form a larger complex. However, the acid-treated Aldolase shows the
single molecule of Aldolase that more directly reflects a single molecule of Aldolase.
Determined molecular weights of aldolase were quite similar in SDS-PAGE and
sedimentation velocity of the acid treated aldolase. It is because these two processes have
denaturing effect in common. SDS can denature proteins by saturating protein with
negative charged SDS, and acidic conditions may cause denaturation by breaking the
internal bonds due to enormous attraction between the H+/OH- of acid/base and the
groups on the surface of. The sedimentation velocity molecular weight determination of
native aldolase is fairly similar to the HPLC determination of molecular weight since the
proteins are not denatured in this method. The molecular weights of aldolase in
denaturing conditions are about one third (1/3) to one forth (1/4) of weights of the native
aldolase. This supports that aldolase is a tetramer. The accepted molecular weight of
aldolase is 40,000 Da for each subunit and 161,000 ± 3,000 Da for the tetrameric protein.
From these previous findings, it would seem that SDS-PAGE is the best method for
determining the molecular weight of monomeric subunits while sedimentation velocity is
the best method for determining the molecular weight of intact, tetrameric aldolase. (2),
(3)
Lysozyme was crystallized in the presence of NaCl and NaI under pH values of 5, 7 and
9 of varying concentrations of 2, 4, 6, 8, 10, and 12%. The crystals formed using NaCl
were tetragonal while those formed with NaI were orthorhombic. These results are
consistent with literature values. The conditions that seemed to be the best were NaCl at
pH 9 of 2 and 4 %, and pH 7 of 8% for NaI. These conditions reflect the best conditions
necessary to optimal growth, most clear crystals possible.(1)
Chemical Characteristics
The isoelectric focusing (IEF) gel in Image 2 shows that purified aldolase was present in
our sample. Lane 6, our sample, has a band that is almost same distance from the sigma
aldolase, which is in the circle in Lane 4. Also, the only protein present in the sample is
aldolase because not other bands or smears are visible of the gel. If there are other bands,
it probably would be proteins of similar size, solubility, and binding specificity as
aldolase that had been purified with the sample. However, their difference in pI value
makes them separate by IEF gel. Also, the charge of a protein can be determined be IEF
at different pH levels. Since the pI of aldolase is 8.17, the net charge of the protein would
be positive at aldolase’s physiological pH of 7.4. The protein will migrate towards the
cathode.
In C terminus analysis, the C- terminal amino acid was found to be tyrosine. The
American Association for the Advancement of Science reported that the C-terminal
amino acid is in fact tyrosine 4. This supports our finding of C- terminal amino acid. The
spots of the different time points on TLC plate, the red color gets more intensity (vivid)
as the time points increase. Therefore, tyrosine at the C-terminal end is important in
aldolase activity because the exposure to carboxypeptidase cleaves the tyrosine.
Also, the color of time point 5 min and time point 15 min are similar, indicating the
exsicion reached a plateau. At a time of 5 minutes, the almost all of Aldolase in sample
had tyrosine excised. Also at 15 minutes, the sample showed a near 100% excision.
Thus, 5 minutes time point is the most affected by carboxypeptidase activity due to the
near complete reduction in Aldolase activity. Therefore, the most effective time point for
C-terminus amino acid determination is at 5 minutes.
References:
1. Petrova, E., P. Dold, and K. Tsukamoto. "Growth of Tetragonal Lysozymecrystals from
Solutions Containing NaCl, CsCl and NaNO3." Journal of Crystal Growth 304.1 (2007):
141-49. Web.
2. Sia, C. and Horecker, B., "The molecular weight of rabbit muscle aldolase and the
properties of the subunits in acid solution." Arch. Biochem. Biophys., v. 123, 186 (1968).
3. Szuchet, S. and Yphantis, D., "Equilibrium sedimentation of proteins in acid solutions.
Dissociation of aldolase by aqueous acetic acid." Biochem., v. 12, 5115 (1973).
4. Koida, M., C. Lai, and B. Horecker. "Subunit Structure of Rabbit Muscle Aldolase:
Extent O# Homology of the A! and /3 Subunits and Age-Dependent Changes in Their
Ratio."ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 134 (1969): 623-31.
ScienceDirect. Web.
5. "Fructose-bisphosphate Aldolase, EC 4.1.2.13." Glycolysis.Co.UK. 21 Nov.
2011. Web. <http://glycolysis.co.uk/aldolase.php>.
Tables and Figures
Image 1. SDS-PAGE Gel of fractions collected from the Purification procedure of
Aldolase.
Log of Molecular Weight (kDa)
SDS- PAGE Standard Curve
2.5
y = -0.3854x + 2.0129
R² = 0.8704
2
1.5
1
0.5
0
0
0.5
1
1.5
2
2.5
Band Distance Traveled (cm)
3
Figure 1 SDS-PAGE standard curve. SDS-PAGE standard curve was generated by the
distance traveled and molecular weights of the Rainbow Marker proteins, and it used for
determination of the molecular weight of aldolase subunits.
Log of Molecular Weight (kDa)
HPLC Molecular Weight Sandard Curve
6
5
y = -1.9895x + 8.0292
R² = 0.9962
4
3
2
1
0
0
0.5
1
1.5
2
Normalized Elution Volume (Ve/Vo)
2.5
Figure 2. HPLC Molecular Weight Sandard Curve. The enzymes used to create the
standard curve are Cytochrome C, Bovine Serium Albumin (BSA), and ß-amylase. The
log of the known molecular weights was taken to generate a linear relationship between
molecular weight and normalized elution volume.
Figure 3. Sedimentation Velocity of Native and Acid Treated Aldolase. The
sedimentation velocities of native and acid treated aldolase sample were taken at multiple
time points. The distance to the peak and adding 5.7 cm first determined the peak
distance. Then the log of the peak distance was determined to create a linear relationship
with time. The slopes of graph were used in the determination of Sobs for native and acid
treated aldolase.
Native Aldolase
Slope
0.0007
Sobs (seconds)
6.812 x 10-13
S20,w (Svedbergs)
7.908 x 10-13
Acid Treated Aldolase
0.0002
1.946 x 10-13
2.259 x 10-13
Table 1. Sedimentation Velocity Data. This table represents the Sedimentation
Coefficients for acid treated and native aldolase. These values were determined with the
slope of the sedimentation graphs .
Image 2. IEF gel of Peak fractions. Our sample is in lane 6. Lane 4 represents a standard
aldolase sample. Lane 5 contains the pI standard markers. The aldolase band can be seen
circled in lane 4.
Figure 4. pI Standard Curve. The graph was generated by using the standard pI values
and the distances of the bands corresponding to the standards.
Molecular
Method
Weight (Da)
SDS-PAGE –Dissociated
42,413
HPLC –Native
116,103
Sedimentation –Acid Treated:
Dissociated
62,306
Sedimentation - Native
158,781
Table2: Molecular Weight of Aldolase from Three Different Methods. This table show
the molecular weight of monomeric or tetrameric aldolase as determined by each method.
Image 3. Alsolase activity after caboxypeptidase exposure.