1. The Purification and
Characterization of Cellular
Retinol Binding Protein from
Bovine Liver Resulting in the
Purification of Catalase
Kimberly Ospedale, Nicole Jonke,
Rohit Singla, and Donald Wunder

2. Cellular retinol binding protein, CRBP, carries retinol throughout the body. In the
experiment, bovine liver was used to purify and characterize this protein. The liver was
homogenized, centrifuged, titrated, applied to a CM-cellulose cation exchange, UNO Q-
12 exchange column, dialysis, and gel filtration. In the characterization process, the SDS-
gel electrophoresis showed a faint and disappearing band around 15,000 g/mole, which
corresponds to CRBP. A final, intense band at a molecular weight of 61,560 g/mole
corresponds to catalase. TCA was found to degrade the catalase, where as methanol had
little to no effect. The reaction of the product with 30% hydrogen peroxide also proved
that the final product is catalase. Although, there was absorption at 350 nm, correlating
with cellular retinol binding protein, the max absorption was at 414 – 410 nm. The
temperature assay found the optimal temperature to be 59˚C, and the pH assay found the
optimal pH to be at 7.75. This data corresponds to that of catalase. The absorption of
fraction 1 of the UNO Q-12 exchange column at 350 nm was not recorded. A decrease in
absorbance from fraction 2-4 indicates that absorption at fraction 1 was larger. This peak
was ignored due to the prominent peaks at fractions 11 and 14. At this point, CRBP was
lost, and catalase became the major protein. The final product contained 202 mg. The
rare, anti-parallel beta barrel composition of CRBP is only shared with one other
molecule, which is catalase.

3. Cellular retinol binding protein, which is produced in the liver, binds and
transports retinol with high affinity and specificity. Retinol is carried in vivo by the
protein. It is also involved in many vitamin A actions (1). Only small amounts of vitamin
A are required daily. Retinol binding protein assures for efficient movement of these
compounds (1). The high affinity prevents the loss of these important molecules. The
protein is found within the cytoplasm of the cells, where it is a member of the lipocalin
family. The protein binds to its carrier protein, which is transthyretin (1). This complex
can interact with certain receptors to deliver retinol to the proper locations. The domain
of the protein consists of a beta-barrel structure, which accommodates the hydrophobic
ligands in the interior (2). The overall structure is also hydrophobic; this causes it to be
associated to lipid-rich areas. Bovine cellular-retinol binding protein has a molecular
weight of 15,823 g/mole. The pI of the protein is 4.92. The quaternary structure contains
just the monomer.
The source of the protein is bovine liver. First, the liver was homogenized. The
protein was titrated and centrifuged several times which removed unwanted debris. The
solution underwent a CM-cellulose cation exchange and filtration. This helped remove
unwanted positively charged proteins, since the binding protein is negatively charged.
The addition of retinol formed a complex with the binding protein, which ensured
stability. The sample was dialyzed to remove unwanted materials. It was submitted for an
FPLC UNO Q-12 exchange cellulose column, which allowed for controlled speed to
better purify the sample. Lastly, a gel-filtration was carried out on a very small sample.
This helped provide very precise data.

4. The goal was to purify cellular retinol binding protein from bovine liver. The
protein was characterized by SDS gel electrophoresis and testing the absorption spectra.
In the SDS gel electrophoresis, only a single band should be observed indicating a single
polypeptide chain. This band indicated the exact molecular weight of our product. In the
absorption spectra, peaks at both 280 nm and 350 nm are expected.
Methods
Purification of cellular retinol binding protein 1 (CRBP1): First, 100 grams of
bovine liver were cut into chunks on ice. The chunks were blended with 200 mL of 0.01
M tris/HCL, pH 7.5, until a smooth consistency was reached. The mixture was then
centrifuged for 20 minutes at 20,000 x g. The supernatant was then poured off and
titrated to pH 5 with glacial acetic acid as it was stirred on a stirring plate. Again it was
centrifuged for 15 minutes at 20,000 x g.
To equilibrate the CM-cellulose, the base of a 400 mL beaker was covered with
CM-cellulose. It was then filled to 225 mL with 0.01 M sodium acetate, pH 5, and
allowed to sit. The supernatant was then stirred in for 30 minutes while refrigerated. The
added cellulose to liver ratio was 1:10 (v/v). The solution was then filtered using a sterile
water aspirator. The filtered solution was then titrated drop wise to pH 7.2 with 6 M
NaOH. Due to a strange coloration, the solution was titrated back to a pH of 6 with 6 M
HCl. Next, 0.250 ul of a 1 mg/mL solution of retinol in ethanol was added, followed by
0.003 g of PMSF, phenylmethylsulfonly fluoride. The solution was centrifuged again at
20,000 x g for 20 minutes. After, the supernatant was poured off and was ultracentrifuged
at 50,000 x g for 120 minutes.

5. The concentration of the protein sample was determined from the standard
Bradford assay. First, the standard curve was made by adding different combinations of
water, bovine serum albumin and the biorad reagent. The following combinations were
used: (Tube 1: 0 BSA/800 water, 2: 200 BSA/ 600 water, 3: 400 BSA/ 400 water, 4: 600
BSA/ 200 water, 5: 800 BSA/ 0 water). 200 microliters of the biorad reagent were added
to each. After 5 minutes, the absorption at 595 nm was measured, recorded, and plotted to
obtain the standard curve. To test our sample, a 10% protein solution was made with 2 ul
of protein and 18 ul of water. 5 ul of this solution was added to 795 ul of water, and 200
ul of the biorad reagent. After the 5 minute waiting period, the solution was tested at a
wavelength of 595 nm, this point was then placed on the standard curve.
The supernatant from the centrifugation was then dialyzed. It was slowly poured
into the dialysis bag. The bag was tied and submerged in a graduated cylinder filled with
0.05 M tris/acetate, pH 8.3. The solution was allowed to dialyze overnight in the
refrigerator with a stirring bar. After the dialysis was complete, the bag was cut and the
solution was submitted for FPLC analysis.
The solution was applied to a UNO Q-12 exchange column (1.5 cm x 15 cm). It
was washed with 150 mL of 0.05 M tris/acetate, pH 8.3. It was then eluted with a linear
gradient of NaCl from 0-0.1 M. Next, the FPLC fractions were analyzed at retinol
binding proteins wavelength of 350 nm. The high absorbance fractions were combined.
They were then applied to a vivaspin concentrator, with a molecular weight cut off at
10,000 daltons, at 2,800 x g for 10 minutes intervals in a swinging bucket rotor for a total
of 3 hours.

6. The concentration of the protein sample was determined from the standard
Bradford assay. The same standard curve was used as prior in the procedure. A 10%
protein solution was made with 2 ul of protein and 18 ul of water. 5 ul of this solution
was added to 795 ul of water, and 200 ul of the biorad reagent. The solution was tested at
a wavelength of 595 nm, then plotted on the standard curve. Next, 0.250 ul of a 1 mg/mL
solution of retinol in ethanol was added.
The sample was then submitted for gel-filtration column (2.5 cm x 50 cm) of
sephadex G-50, eluted with 25 mM PO4 and 0.3 M KCl, pH 7.2. The fractions from the
gel-filtration were tested in the spectrophotometer at a wavelength of 350 nm. The
fractions with the highest absorbance readings were combined (1). The absorbance at 350
nm was also taken for each of the 6 protein samples.
The concentration of the protein sample was determined from the standard
Bradford assay. Here, a new standard curve was made, following the same procedure as
stated previously. The amount of protein added, absorption reading, micrograms of
protein present and concentration is shown in table 3.
To prepare for the SDS-gel electrophoresis, the samples taken periodically
throughout the procedure were diluted to a concentration of 2 mg/mL. The samples were
diluted again by adding 20 ul of the protein solution to 20 ul of the sample application
buffer in eppendorf tubes. The sample application buffer contained glycerol, blue dye,
and beta-mercapto-ethanol. They were heated in boiling water for 10 minutes, and then
quickly centrifuged. 25 ul of the samples were added to each well. 10 ul of the molecular
weight standard solutions were also added to the last well. The gel was run at a constant
200 V for 30 minutes. After the power was turned off the gel plate was removed from the

7. buffer chamber. The gel plate was then cracked open exposing the gel, which was
carefully placed in the staining solution. The staining solution contained 0.25%
coomassie blue in methanol: Acetic acid: water (5:1:5). After 15 minutes, the staining
solution was removed, and destaining solution was added and left for 15 minutes. The
destaining solution contained acetic acid: methanol: water (7:7:86). The destaining
solution was left over night. When the gel was observed, there was an unknown band at a
molecular weight of 50,000. Due to this strange band, the absorption of each sample was
tested at a range of wavelengths.
To further test the contents of the final sample, trichloroacetic acid was used to try
and denature the protein. TCA analysis was completed. 500 ul of the protein sample and
500 ul of TCA were combined in an eppendorf. After 10 minutes a precipitate formed.
The solution was then centrifuged in a cold environment for 10 minutes at 14,000 rpm.
The supernatant was then poured into a cuvette and the absorbance of the sample was
tested at a range of wavelengths.
In addition to denaturing our protein, 500 ul of methanol was combined with 500
ul of our final protein. The solution was then centrifuged in a cold environment for 10
minutes at 14,000 rpm. The supernatant was then poured into a cuvette and the
absorbance of the sample was tested at a range of wavelengths.
The final product was thought to contain catalase. To test this theory, 1 mL of
buffer, 50 ul of protein, and 10 ul of 30% hydrogen peroxide were combined. Since a
reaction occurred, an additional hydrogen peroxide assay was done. 985 ul of 0.5 M
tris/acetate, pH 8.3, 5 ul of hydrogen peroxide, and 10 ul of the protein sample were
combined. 10 minutes were allowed for the reaction to be complete. The sample was then

8. allowed to vortex for 1 minute to get rid of any bubbles, and centrifuged for 2 minutes at
12,000 rpm. The sample was tested at a wavelength of 240 nm, which is the absorbance
for hydrogen peroxide. The concentrations found for each of the 6 samples were then
multiplied by 0.01 mL, the amount of protein used for the assay, to give the mg of the
protein. The absorbance for each of the samples at 240 nm was then divided by the mg of
protein in each sample. This gives the specific activity of each sample of catalase.
The 6 protein samples, as well as catalase, were applied in an SDS-gel electrophoresis
following the same procedure as above. The final sample was then submitted for H1
NMR analysis. To prepare for the NMR, one sample was made with 900 ul of 0.05 M
tris/acetate, pH 8.3, and 100 ul of D2O. Another sample was made with 100 ul of the final
protein sample, 100 ul of D2O and 890 ul of water. A third sample was made with 895 ul
of 0.05 M tris/acetate, pH 8.3, 5 ul of H2O2, and 100 ul of D2O. The last sample contained
885 ul of 0.05 M tris/acetate, pH 8.3, 5 ul of H2O2, 10 ul of the final protein sample and
100 ul of D2O. The samples were left alone for 10 minutes to react, vortexed for 1
minute, and centrifuged for 2 minutes at 12,000 rpm. 610 ul of each sample were
submitted for NMR.
To further characterize our final protein, the effect of pH on catalase was tested.
200 ul of the sample, 5 ul of H2O2, and 795 ul of different pH buffers were combined.
The buffers ranged from 4-10, and increased in 0.25 intervals. Another group previously
prepared these buffers. The solutions were allowed to react for 10 minutes. They were
then vortexed for 1 minute, and centrifuged for 2 minute at 12,000 rpm to get rid of any
bubbles. The absorbance was then recorded for each sample at 240 nm.

9. Another characterization test was testing the effect of temperature on catalase
activity. To do so, 200 ul of the final product, and 795 ul of the neutral pH buffer were
combined. They were then placed in different hot baths of varying temperatures for 30
minutes. The temperatures that were tested were 40˚C, 45˚C, 55˚C, 60˚C, 65˚C, and
80˚C. The temperature was controlled by adding ice when necessary. After the 30 minute
period, the solutions were allowed to cool. Then, 5 ul of H2O2 was added. The solutions
were then allowed to react for 10 minutes. They were vortexed for 1 minute, and
centrifuged for 2 minutes at 12,000 rpm to get rid of any bubbles that may diffract the
light. The absorption at 240 nm was then taken for each sample.
Results
Homogenization of the bovine liver produced an opaque, light red solution. After
centrifugation, both a soft and hard white pellet was produced. The supernatant, which
was kept, retained a dark pink color. The pellet was a much lighter pink. After the
titration to pH 5 with glacial acetic acid and second centrifugation, the solution was a
transparent, bright red.
The volume obtained at this point was 145 mL, so 14.5 mL of CM-cellulose was
added. After the titration to pH 7.2 with 6 M NaOH, the solution appeared to gain its
opaque, light pink color, and a precipitate began to form at the bottom layers. This was
the reason for titrating it back to a pH of 6. After centrifuging, and ultra centrifuging the
solution and removing the pellets, the bright red color returned. During the Bradford
assay, the sample gave an absorbance reading of 0.214. In matching this point on the
standard curve, the concentration was found to be 10.05 (Figure 1).

10. 100 mL of the protein solution was applied to a UNO Q-12 exchange column (1.5
cm x 15 cm). The FPLC column produced a protein profile with 3 major peaks at
fractions 1, 11 and 14 (Figure 2). At this point, the majority of the fractions had a light
brown color. Fraction one still maintained a bright red color. The elution position was
around 260 mL of the total 500 mL gradient. This data agrees with previous data. The
absorbance at 350 nm was determined for each fraction to determine the peaks with the
most cellular retinol binding protein activity (Table 1). Fractions 11-15 were combined
since they had the highest absorption readings.
During the Bradford assay, the sample gave an absorption reading of 0.113. In
matching this point on the standard curve, the concentration was found to be 4.949
(Figure 1). 5 mL of the protein solution was the submitted for the gel-filtration. The gel-
filtration column produced a protein profile with 1 major peak at fraction 9 (Figure 3).
The fractions from the gel-filtration were tested in the spectrophotometer at 350 nm
(Table 2). Fractions 7-10, with the highest absorptions, were combined.
A Bradford assay was then completed on each of the 6 samples. The amount of
protein added, absorption reading, micrograms of protein and concentration is shown in
table 3. The micrograms of protein added and their concentrations was plotted on the
Bradford standard curve (Figure 4) The absorption of these samples at 350 nm is
recorded in Table 4.
Each sample was then submitted for a SDS-gel electrophoresis, the gel is shown
in figure 5. There is a band around a molecular weight of 15,000 g/mole that disappears
after the 4th sample. At a molecular weight just above 50,000, there is a band that

11. becomes darker after each sample. Figure 6 shows the graph used to determine the exact
molecular weight of the band, this was calculated to be 61,560 g/mole.
In the attempt to characterize this dark band, each sample was tested at a range of
wavelengths; this data is presented in table 5. Generally the max absorption of the
samples was between 404 nm and 410 nm. In the TCA analysis, there was no longer a
max at 410 nm; the color here was destroyed (figure 7). In the methanol analysis, the max
absorption of 0.121 remained at 410 nm, with little protein destruction (figure 8). In
testing final product with a small portion of hydrogen peroxide, the reaction produced
bubbles immediately upon addition. In testing each of the 6 samples with hydrogen
peroxide, they each produced bubbles immediately upon addition. As more catalase was
present in each sample, more hydrogen peroxide was degraded therefore lowering the
absorption reading. Through the purification process, the absorption readings decreased
indicating more catalase is present (table 6). Also, throughout the purification process,
the specific activity decreased indicating more catalase is present (table 6).
Figure 9 shows the second SDS-gel electrophoresis that was complete using pure
catalase. Although the pure catalase appears as 2 bands, its upper band directly lines up
with the final sample in lane 6. Figure 10 shows the graph used to determine the exact
molecular weight of the band, this was calculated to be 64,879 g/mole. The upper band of
the pure catalase was also calculated to be 64,879 g/mole.
The data from the NMR analysis is shown in figure 11. We found that the
hydrogen peroxide resonated too close to the water to get desirable results. The effect of
pH on catalase activity is shown in figure 12. The peak at a pH of 4.75 indicates a lot of
hydrogen peroxide is present, and therefor no catalase. The minimum at 7.75 indicates no

12. hydrogen peroxide is present, and here is the optimal pH for catalase. The effect of
temperature on catalase activity is shown in figure 13. There is a minimum point on the
curve just below 60˚C; this is the optimal temperature for our sample. Any temperature
higher than 60˚C degrades catalase and looses all activity.
Discussion
The SDS-gel electrophoresis shows that there was a band at a molecular weight of
15,000. This band is consistent with the molecular weight of cellular retinol binding
protein, which is 15,823 g/mole. After sample 4, this band disappears almost completely.
At this point in the procedure, a band that was present around a molecular weight of
50,000 g/mole becomes very intensified. Calculations provide the exact molecular weight
to be 61,560. This very closely matches the actual molecular weight of catalase, which is
59,900 g/mole. Catalase degrades hydrogen peroxide. It is evident that catalase is present
when each of the 6 samples react immediately upon the addition of hydrogen peroxide.
There were some absorption peaks at 350 nm, which corresponds to the absorption of
cellular retinol binding protein. When the samples were tested at a range of wavelengths,
the true max was found between 404 – 410 nm. This max corresponds to the absorption
of catalase (4). The data shows that throughout the procedure catalase was present and
becoming concentrated. The final product of catalase was 40 mL. This had a
concentration of 5.05 mg/mL, making the total protein isolated 202 mg. After the UNO
Q-12 exchange column, most fractions appeared as a light brown color. This color
matches the appearance of pure catalase. It is evident that at this point that the main
protein present is catalase. The absorption of fraction 1, which appeared red, of the UNO

13. Q-12 exchange column at 350 was not calculated. A decrease in absorbance from fraction
2 and 4 indicate that absorption at fraction 1 must have been larger. This peak was
ignored due to the major, prominent peaks at fractions 11 and 14. At this point, cellular
retinol binding protein was lost, and catalase became the major protein present. Research
states that the only other molecule besides cellular retinol binding protein, with the rare 8
stranded, anti-parallel beta barrel is catalase (1). The pH analysis gave an optimum pH of
7.75, similar to the accepted literature value is 6.8-7. The temperature analysis gave an
optimum temperature of about 59˚C. This point is close to the accepted literature value of
55˚C (1).

14. References
(1) Newcomer, M. E., Jones, T. A., Aqvist, J., Sundelin, J., Eriksson, U., Rask, L., &
Peterson, P. A. (1984). The three-dimensional structure of retinol-binding protein.
The EMBO journal, 3(7), 1451.
(2) Ong, D. E., & Chytil, F. (1978). Cellular retinol-binding protein from rat liver.
Purification and characterization. Journal of Biological Chemistry, 253(3), 828-
832.
(3) "UniProtKB - P02694 (RET1_BOVIN)." Uniprot. 4 Oct. 2015. Web. 8 Nov. 2015.
(4) Walton, P. A., & Pizzitelli, M. (2012). Effects of peroxisomal catalase inhibition on
mitochondrial function. Frontiers in physiology, 3.

15. Figure 1: The standard bradford assay curve for FPLC and gel filtration.
Fig.2. FPLC- Chromatography of CBP1 on Uno Q-12 exchange column (1.5 cm x 15
cm). The protein washed with 150 mL of 0.05 M tris/acetate, pH 8.3. The protein was
then eluted with a linear gradient of NaCl from 0-0.1 M.
y = 0.0396x + 0.015
R² = 0.98283
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 5 10 15
A595
Protein (ug)
Series1
Linear (Series1)

16. Table 1: FPLC Uno Q-12 exchange column fraction analysis. The bolded fractions were
the combined fractions.
Fig.3. Gel-filtration of CBP1. The protein solution was applied to a 2.5 cm x 50 cm gel-
filtration column of sephadex G-50. It was then eluted with PO4 and 0.3 M KCl, pH 7.2.
The green line represents the absorbance at 280 nm.
Fraction Reading
17 0.238
18 0.169
20 0.084
22 0.055
24 0.039
26 0.034
28 0.034
29 0.032
30 0.090
31 0.128
32 0.029
33 0.024
Wash 0.981
Fraction Reading
Blank 0.000
2 0.136
4 0.011
6 0.008
8 0.014
9 0.022
10 0.171
11 0.523
12 0.482
13 0.510
14 0.556
15 0.403
16 0.326

17. Table 2: Gel-filtration fraction analysis. The bolded fractions were those combined.
Table 3: The amount of protein added to the Bradford assay solution, their absorbance
reading, the amount of micrograms of protein present and their concentration.
Sample
Amount of
protein added
(ul)
Absorbance
reading
Micrograms
of protein
mg/mL
1 0.125 0.206 3.42 27.36
2 2.5 0.346 7.10 2.844
3 1 0.449 9.82 9.82
4 1 0.338 6.89 6.89
5 1 0.269 5.08 5.08
6 1 0.268 5.05 5.05
Figure 4: The standard bradford assay curve for SDS-gel electrophoresis
Fraction Reading
2 0.033
3 0.048
4 0.073
5 0.063
6 0.075
7 0.116
8 0.338
9 0.474
10 0.293
11 0.089
12 0.047
13 0.033
Fraction Reading
14 0.038
15 0.044
16 0.043
17 0.014
18 0.024
19 0.025
20 0.048
21 0.026
22 0.016
23 0.018
24 0.008
25 0.012

18. Table 4: The absorbance at 350 nm of the 6 samples being submitted for SDS-gel
electrophoresis.
Sample
Amount of
protein added
(ul)
Absorbance
1 10 0.406
2 40 0.133
3 50 0.161
4 100 0.276
5 200 0.155
6 1000 0.318
Lanes 1 2 3 4 5 6 7
75
50
37
25
20
15
10
Figure 5: shows the SDS-gel electrophoresis. Lanes 1-6 represents the 6 samples of
protein. Lane 7 shows the molecular weight standards.

19. Figure 6 shows the log of the molecular weights that make up the molecular weight
standards ladder vs. their migration in cm.
Table 5 shows the max wavelength for each sample tested at a range of 400 nm-740 nm
A B
Figure 7 A shows the reading of sample 6 over a range of wavelengths from 350-700 nm.
Figure 7 B shows sample 6 over the same range of wavelengths after the addition of
TCA.
Sample
Max wavelength
(nm)
1 407
2 410
3 410
4 410
5 404
6 404

20. A B
Figure 8 A shows the reading of sample 6 over a range of wavelengths from 350-700 nm.
Figure 8 B shows sample 6 over the same range of wavelengths after the addition of
methanol.
Table 6 shows the absorption reading at 240 nm and the calculated specific activity for
each sample, as well as pure catalase.
Samples A240
Specific
Activity
1 -2.502 -9.141
2 -2.842 -100.0
3 -2.900 -29.54
4 -2.865 -41.55
5 -2.985 -58.77
6 -2.990 -59.17
Pure
catalase
-3.053 -152.7

21. Lanes 1 2 3 4 5 6 7 8
Molecular Standards 75
50
37
25
20
15
10
Figure 9 shows the SDS-gel electrophoresis. Lanes 1-6 represents the 6 samples of
protein. Lane 7 shows pure catalase. Lane 8 shows the molecular weight standards.
75
50
37
25
20
15
10
1 2 3 4 5 6
Figure 10 shows the log of the molecular weights that make up the molecular weight
standards ladder vs. their migration in cm.

22. Catalase
Figure 11 shows the data received from NMR. The first row is H2O2 and catalase. The
second row is H2O2. The third row is catalase. The last row is just the buffer.
Figure 12 shows the pH vs. absorbance at 240 nm.

23. Figure 13 shows temperature (˚C) vs absorbance at 240 nm.