1. Figures 3A- 5B. Characterization and purification of hTyr protein from chromatography. Figures 3A and 3B represent
chromatograms obtained from the afinity purification using a HisTrap Crude affinity column with 0.1% Triton X-100 and 5 mM
Fos-cholnie-12 respectively. Both contained Triton X-100 in the binding buffer and the respective detergent in the elution
buffer. Figures 4A and 4B represent the first size exclusion chromatography with a Sephacryl S200 (120 mL) column, while
Figures 5A and 5B represent the second size exclusion chromatography with a Superose 12 (24 mL) column. Within the figures
is a SDS-PAGE (1), Western Blot (2), and activity assay (L-dopa assay, 3) corresponding with the labeled fractions above. Boxed
regions refer to the fractions collected for future analysis.
Purification and biochemical characterization of
full-length human tyrosinase
Nicole Kus, Monika Dolinska, Yuri Sergeev
The human tyrosinase (hTyr) is a Type 1 membrane protein with an alpha helix spanning
the membrane of the melanosome. Tyrosinase is a glycoenzyme which contains seven N-
glycosylation sites that help maintain the thermodynamic stability and functionality of the
protein. With two copper ions in the active site, tyrosinase is the key enzyme involved in
melanogenesis- the production of melanin. Namely, it is able to catalyze the initial and rate-
limiting steps of the hydroxylation of L-tyrosine into L-dopa and the oxidation of o-diphenol
into L-dopaquinone. About ~300 mutations in the hTyr gene (3kb) are associated with
oculocutaneous albinism type 1 (OCA-1), an autosomal recessive disorder. Previously, a
truncated tyrosinase (hTyrCtr) was created which lacked the trans-membrane helix (1). The
hTyrCtr was successfully purified in our lab and existed in a monomeric state
with a molecular weight of approximately 56,000 Da.
Purpose
Purify a glycosylated and functionally active membrane bound tyrosinase (hTyr) using
detergents to solubilize the protein. Characterize the activity, oligomeric state of the protein,
and enzymatic abilities (Km, Vmax, and kcat).
Tyrosinase
Tyrosinase
L-tyrosine
L-dopa
Dopachrome
OCA Type 1A OCA Type 1B
Tyrosinase
Purification
Testing
different
detergents on
infected T. ni
larvae
Homogenize
larvae (1.0%
Triton X-
100)
Sonicate,
centrifuge,
and filter
sample
Crude HisTrap
Affinity Column
Size Exclusion
Column
Characterize
Protein &
enzymatic
activity
assays
Methods
Figure 1. First two steps of the
melanin production pathway.
Figure 2. Methods used for tyrosinase purification.
Results: Protein Identification Results: Enzyme Kinetics
Ophthalmic Genetics and Visual Function Branch, NEI
Future Work
Triton X-100 Fos-Choline-12
34 35 36 37 38 39 40 4134 35 36 37 38 39 40
41
Affinity (HisTrap FF 5 mL)
1
2
3
1
2
3
16 17 18 19 20 21 22 23 2416 17 18 19 20 21 22 23
Km (mM) Vmax (mOD min-1) kcat (sec-1)
hTyr 0.67 ± 0.02 4.49 ± 0.12 140.04 ± 28.88
hTyrCtr 0.74 ± 0.04 9.84 ± 0.33 103.23 ± 16.26
Size Exclusion #1 (Sephacryl S200 16/60)
Peak molecular weight ~ 100 kDa
Size Exclusion #2 (Superose 12 10/300)
17 18 19 20 21 22 23 24 25 26 27 28 29 30
• Test more detergents to determine which is best for purification.
• Purify more membrane bound tyrosinase for further characterization.
• Conduct sedimentation equilibrium to determine the oligomeric state of
the protein.
• Conduct atomic force microscopy to determine radius of tyrosinase.
• Perform kinetic assays of the protein in Fos-Choline-12 detergent to compare
with results in Triton X-100.
• Run kinetic assays in the presence of known inhibitors and activators of
tyrosinase.
• Improve purification protocol to obtain a larger quantity of protein.
• Improve purity of protein obtained.
Discussion
Conclusions
• Fos-Choline-12 and Triton X-100 were useful detergents for purifying tyrosinase.
• In Triton X-100, tyrosinase appears to have a molecular weight of 113,000 Da.
• In Fos-Choline-12, tyrosinase has an apparent molecular weight of ~100,000 Da.
• In contrast to SEC, which suggested a dimeric state for hTyr, sedimentation equilibrium
shown hTyr as a monomeric molecule (62 kDa).
• The difference between SEC and the sedimentation equilibrium could be attributed to
the interactions of glycosylated sugars, or the result of micelle formation and residual
lipid interactions. These interactions could possibly increase the apparent molecular
weight on the size exclusion chromatogram.
• The Michaelis-Menten constant, Km, shows a similarity between the truncated and full
length protein (0.74 mM and 0.62 mM respectively).
• As the C-terminus is lacking in hTyrCtr, but the enzymatic activity is similar to that of
hTyr, this shows that the C-terminus is important in localizing tyrosinase to the
membrane but not in enzymatic function.
• Triton X-100 absorbs in the UV spectra and interfers with determining protein
concentration.
Specific Activity
hTyr 238,741 ± 6,370
hTyrCtr 426,603 ± 18,736
Table 1. Michaelis-Menten constants comparing the purified membrane bound protein
to the purified truncated protein.
Table 2. Specific activity calculations for the
purified membrane bound protein (hTyr) and
the purified truncated protein (hTyrCtr). Figure 6. Michaelis-Menten kinetics
Membrane proteins play essential roles in maintaining cellular and tissue function. They
account for over 50% of drug targets and approximately 30% of protein encoding genes
encode for membrane proteins. Although they are incredibly important, there is a lack of
information regarding membrane proteins due to a difficulty of purification, as they are
not soluble in aqueous solutions. In order to solubilize membrane proteins, it is
essential to use a detergent which can shield the hydrophobic transmembrane domains.
Tyrosinase, a Type I membrane bound protein located in the melanosome, contains an
alpha helix which spans the membrane. It functions as the key enzyme in melanogensis,
the production of melanin, by playing a role in the initial and rate-limiting steps.
Mutations in tyrosinase have been linked to a variety of disorders, namely
oculocutaneous albinism type 1 (OCA1) and hyperpigmentation. Thus, purification of
the full-length human tyrosinase is essential to understanding the biochemical
properties, which can aid in finding a new drug that can either improve or decrease its
function. In purifying the full-length protein, this project hopes to find a suitable
detergent to use for characterization of the oligomeric state and activity of the protein.
- No pigmentation
- Inactive tyrosinase
- Blue/translusent iris
- White hair, eyebrows,
and skin
- Visual problems
- Decreased pigmentation
- Reduced tyrosinase
activity
- Light skin color which
can darken
- Light to dark hair color
- Visual problems
References
1. Dolinka, M., Kovaleva, E., Backlund, P., Wingfield, P.T., Brooks, B.P., Sergeev, Y.V.
“Albinisim-causing mutations in recombinant human tyrosinase alter intrinsic
enzymatic activity.” PLOSONE. January 2014.
Introduction
Columns used:
- HisTrap Crude 5 mL
- Sephacryl S200 16/60
- Superose 12 10/300
Gel Filtration Buffer:
- 50 mM Tris
- 1 mM EDTA
- 150 mM NaCl
- 0.1 % Triton X-100 or
5 mM Fos-Choline-12
IMAC Elution Buffer:
- 20 mM Sodium Phosphate
- 500 mM NaCl
- 500 mM Imidazole
- 0.1 % Triton X-100 or
5 mM Fos-Choline-12
IMAC Binding Buffer:
- 20 mM Sodium Phosphate
- 500 mM NaCl
- 20 mM Imidazole
- 0.1 % Triton X-100
1
2
1
2
3
22
1 1
L-dopa activity assay:
- 10 µL Sodium Phosphate
with 3 mM L-dopa
- 10 µL purified protein
22 23 24 25 26 27 28 29
Results: Sedimentation Equilibrium
Peak molecular weight ~ 113 kDa
5B.5A.
4A. 4B.
3B.3A.
Figure 6. Homology model of human full-
length tyrosinase anchored to the
melanosomal membrane. Trans-
membrane helix is labeled in red.
Figure 7. Graph of sedimentational equilibrium
determining the oligomeric state of tyrosinase.
According to the graph, tyrosinase exists as a
monomer with an approximately molecular weight of
62 kDa. The solid line indicates the theoretical value,
while the circles represent the experimental data.
hTyrCtr
hTyr