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 41

34 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 24

16 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 ActivityhTyr 238,741 ± 6,370hTyrCtr 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

References1. 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.

hTyrCtrhTyr