Synergy of H
2
O
2
and Methionine Affects Tyrosinase Activity, Causing
Repigmentation of Gray Hair
Haiming Sun
Suzhou High School of Jiangsu Province, Suzhou, Jiangsu 215000, China
Keywords:
Methionine, H
2
O
2
, Tyrosinase, Methionine Sulfoxide Reductase a and b, Immunofluorescence, Western Blot,
Hair Follicle Melanocyte, Epidermal Melanocyte.
Abstract: Hair colour demonstrates people’s health condition. The most prevalent explanation for hair graying is that
peroxides oxidize tyrosinase, preventing this enzyme from activating melanocytes. From Schallreuter’s paper
in 2004, hydrogen peroxide was found to activate acetylcholinesterase, the enzyme crucial in treatment of
vitiligo. Vitiligo is supposed to be caused by deactivation of pigment cells, like the possible cause of hair
graying. Meanwhile, methionine has long been considered to eliminate peroxides. Therefore, it’s attractive to
see that if the combination of the two materials, hydrogen peroxide and methionine, will activate tyrosinase
more largely than any one of them do. This research uses the rate of L-Dopachrome formation to determine
tyrosinase activity. There are two possible results. The ideal result is the synergy of methionine and hydrogen
peroxide activates tyrosinase. However, since hydrogen peroxide can deactivate tyrosinase, the two
activations may cancel out so there is no significant change in tyrosinase activity. If synergy of hydrogen
peroxide and methioine do activate tyrosinase better, then the problem of hair graying can be better solved.
1 INTRODUCTION
Methionine (Met) plays a critical role in the
metabolism of the body. It is an antioxidant that slows
down the aging process. Meanwhile, with a high level
of Met in the body, the level of the other amino acids
will also be enhanced, which strengthens the immune
system. Additionally, Met has been confirmed able to
detoxify toxic substances like heavy metals and
prevent liver damage. Therefore, Met has been used
in many health products and is widely accepted
(University of Rochester Medical Center). According
to Peggy Sextius' research, Met extracted from
polygonum multiflorum radix could slow down or
prevent hair graying (Sextius, 2017).
Several explanations are proposed for the gradual
loss of hair pigmentation. Studies in DNA repair have
shown the critical roles of DNA repair, telomerase,
Bcl-2, and stem cell factor for hair pigmentation
[REFs]. One prevalent theory, the free radical theory
of graying, proposes the association between
melanogenesis and tyrosinase activity. J. M. Wood
and others' study further supports the free radical
theory, in which he suggested that H
2
O
2
oxidizes Met
to Met-S=O and inactivates the enzyme catalase
breaking H
2
O
2
down to water and oxygen; and he also
observed that tyrosinase containing Met is inhibited
(Wood, 2009). Thus, it is proved that H
2
O
2
-induced
oxidation of Met directly affects tyrosinase and
hinders follicular melanogenesis.
The study by Ikeda et al. revealed Met is able to
induce tyrosinase production in two different
Streptomyces. The total cellular tyrosinase activity is
induced 16.9-folder higher in the presence of Met
than in the negative control (Ikeda, 1996).
In the study of Karin U. Schallreuter, there's a
return of systemic enzyme activities to normal after
application of a pseudocatalase PC-KUS. The
experiment is in vitro, conducted on skin biopsies
from patients with vitiligo, whose H
2
O
2
concentration
in vivo is identified to be in the 10-3 M range.
Schallreuter et al. observed that the complete removal
of H
2
O
2
by PC-KUS led to increased epidermal
catalase compared to the high concentration control
(10-3 M H
2
O
2
); however, the removal of H
2
O
2
led to
decreased reaction rates compared to the low
concentration control of H
2
O
2
(10-6 M H
2
O
2
)
(Schallreuter, 2004).
While high concentrations of H
2
O
2
exhibit
significant inhibition on the expression of
acetylcholinesterase (AchE), low concentrations of
H
2
O
2
are discovered to activate AchE. The
92
Sun, H.
Synergy of H2O2 and Methionine Affects Tyrosinase Activity, Causing Repigmentation of Gray Hair.
DOI: 10.5220/0012013800003633
In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 92-96
ISBN: 978-989-758-637-8
Copyright
c
 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

understanding of H
2
O
2
's two roles in vitiligo-the
depigmentation disorder can be applied to the hair
graying process as well. With the help of Met to
prevent H
2
O
2
-induced inhibition of tyrosinase, I
predict that increasing amounts of L-Methionine
combined with low amounts H
2
O
2
may maximize the
tyrosinase activity. The negative control is adding no
Met or PC-KUS, one positive control is adding PC-
KUS and the other positive control is adding Met.
2 MATERIALS AND METHODS
All research involving human subjects is approved by
local ethics committees and adhered to the Helsinki
declaration (Wood, 2009).
Hair follicle cells are divided into four groups,
three control groups, and one experimental group,
from face-lift surgery. As a positive control group
(activation), met and grey hair follicle melanocytes
(HFMs) are used (group 1). The H
2
O
2
concentration
in grey and completely white HHFs is determined to
be 10-3 M. Another positive-control group consisted
of grey HFMs supplemented with PC-KUS
(activation) (group 2). The negative control consisted
entirely of grey HFMs with no additives
(deactivation) (group 3). The experimental group
consisted of Met, PC-KUS, and grey HFMs (group 4).
2.1 Cell Culture of HFMs and
Preparation of HHF
Hair follicle melanocytes (HFMs) and human hair
follicles (HHFs) are harvested from normal human
scalp skin obtained from face-lift surgery with gray
or completely white hair (n=2, female; n = 20, 10
female, 10 male respectively). By using a
microdissection microscope, Anagen VI, HHFs are
isolated. Briefly, scalp skin is divided into small
pieces, about 1 cm2 each. From the subcutis, the
dermis is isolated.
2.2 Preparation of HHF and HHF Cell
Extracts
To prevent denaturation, cell extracts are attained
from intact anagen HHFs and cell cultures with the
use of a mini pestle, a mortar (- 80°C), and fine sand.
After 5-minute centrifugation at 7000 g, cell pellets
and HHFs are ground in Tris buffer. The collected
supernatant is aliquoted and stored at - 80°C and is
analyzed by the Dc-protein assay (Wood, 2009).
2.3 In Situ Immunofluorescence
Protein Location in the Separated
HHFs
Isolated HHFs are kept in OCTTM compound
(Sakura, Eastbourne, UK) at -80°C before conducting
experiments with the cryostat as described in previous
studies [REF]. To briefly describe it, 20 frozen slides
were divided into 4 different groups: 5 frozen slides
with 5-μm cryosections of gray HHFs(n=3) and shafts
are added narrowband UVB-activated pseudocatalase
PC-KUS. 5 frozen slides are added extra Methionine
(10×10-3 M). 5 frozen slides are added both PC-KUS
and extra Methionine. 5 frozen slides are added
nothing. The 20 frozen slides are air-dried at room
temperature, settled in ice-cold methanol, and
blocked-in normal donkey serum, finally washed in
phosphate-buffered saline (PBS). Methionine
sulfoxide reductase A (MSRA) is detected through a
polyclonal rabbit anti-human antibody and then
incubated at room temperature. Methionine sulfoxide
reductase B (MSRB) detection utilizes a monoclonal
mouse anti-human antibody (Autogen Bioclear) and
then incubated overnight at 4°C. Following the above,
the slides are washed with PBS, air-dried, and then
incubated at room temperature with a fluorescent
secondary antibody (conjugated donkey anti-rabbit or
anti-mouse) with a wash with PBS for 3 times, air-
dried, and mounted in Vectashield Mounting Medium
containing DAPI (4',6-diamidino-2-phenylindole) in
order to identify specific nuclear. Slides are viewed
under a Leica DRMIB/E fluorescence microscope,
and images are captured with a digital camera, and the
imaging software facilitates the observation of
imported images on the computer. (Wood, 2009)
2.4 Western Blot
According to the description above, gray and
completely white HHF extracts are obtained. Before
loading onto a polyacrylamide gel for protein
separation, sample buffer (10% SDS,
mercaptoethanol, glycerol, and 0.5 M Tris/HCl) is
added to the supernatants. The polyacrylamide gel is
electroblotted onto a PVDF membrane before any
nonspecific binding sites are blocked by immersion at
room temperature of the membrane in a gelatin/ TBS-
Tween buffer blocking solution. After this step, the
primary antibodies are incubated overnight at room
temperature in buffer. The antibodies, rabbit
antiMSRA, rabbit anti-MSRB, mouse anti-catalase,
and goat anti-actin are used. After being washed for
40 min in buffer, the blot is next incubated for 1 h at
Synergy of H2O2 and Methionine Affects Tyrosinase Activity, Causing Repigmentation of Gray Hair
93

room temperature with an antibody listed above.
Using moderated enhanced chemiluminescence
(ECL) fixed on a film sheet, visualization of the
specific protein bands is performed (Wood, 2009).
2.5 Determination of Tyrosinase
Activity
The rate of L-Dopachrome formation, measured by a
spectrophotometer with optical density of 475 nm,
with L-tyrosine as substrate, is utilized to determine
tyrosinase action. The rate of reaction is determined in
a linear period of 2 min. Since 1×10-3 M L-tyrosine
produces 1×10-3 M H
2
O
2
, 3×10-3 M L-tyrosine is
added. Thus, H
2
O
2
is produced with a one-to-one ratio,
that is, 3×10-3 M. The experiments are performed in
the presence of different L-Methionine concentrations
(6×10-3 M) and the amount of PC-KUS added (6×10-
3 M) in groups 1, 2, and 4. All experiments are
conducted twice. Since Met-tyrosinase is activated by
L-Dopa, L-Dopachrome formation is followed and
recorded from L-Dopa at 570 nm. With a microplate
reader, L-Dopachrome will be determined every 2 min
over 15 min (Wood, 2009).
2.6 Determination of MSRA and
MSRB Enzyme Activities
Consistent with the explanation above, from isolated
HHFs for HFMs and other HHFs cells, complete
HHFs extracts are acquired. In brief, the reaction
mixture contains cell extract and [14C] Methionine
sulfoxide, with dithiothreitol (DTT) functioning as an
electron donor. Reactions are incubated for 1h at
room temperature. Then reaction product is applied to
a TLC silica gel plate and graphed by a
chromatographer in “isopropanol: formic acid: water”
(20:1:10). Ninhydrin is used to detect L-Methionine
and Met-S=O. Radiolabeled and scraped from the
TLC plates, [14C] L-Methionine spots are added to
scintillation fluid and recorded on the 14C channel in
a Packard Tricarb Liquid Scintillation Counter. The
formation of L-Methionine is standardized to
mmol·mg-1 ·(0.5h)-1 of protein (Wood, 2009).
2.7 In Vitro and in Vivo FT-Ramen
Spectroscopy for Detection of
H2O2, Met-S=O in H2O2-Oxidized
Tyrosinase and Gray Hair
Human white hair shafts from the scalp hair are cut
into pieces and a FT-Raman spectroscopy is used to
analyze it. A Bruker RFS spectrometer together with
a liquid-nitrogen-cooled germanium detector help
acquire FT-Raman spectra. Near-infrared excitation
is produced by a Nd33+: YAG laser. Each spectrum
is accumulated over with scans and a resolution of 4
cm-1. Detection of H
2
O
2
is by perceiving the O=O
stretch. Met-S=O is visualized too. Tyrosinase
(10×10-3 M) are lyophilized and measured as solids.
3 RESULTS
There is no H
2
O
2
detectable in group 1(HFMs, Met)
while there is a presence of 10-3 M H
2
O
2
concentrations in group 3(HFMs). The presence of
Met-S=O is higher in group 3 than in groups 1 and
2(HFMs, PC-KUS). Results by immunofluorescence
and Western blot analysis show that catalase, MSRA
and MSRB protein expressions are weaker in group 3
than it is in group 1 and group 2. Tyrosinase in group
1(HFMs, Met) shows activity larger than the negative
control group (group 3, nothing added). Tyrosinase in
group 2(HFMs, PC-KUS) shows activity larger than
the negative control group.
Based on Wood et al's study (2009) (Wood, 2009),
H
2
O
2
is assigned at 875 cm-1 based on the O=O
stretch and Met-S=O is assigned at 1030 cm-1. H
2
O
2
oxidized Met to Met-S=O. MSRA and MSRB's
function is to repair oxidized Methionine Met-S=O.
However, aging decreases the two enzymes' level in
the body, MSRA and MSRB are not enough for
repairing Met-S=O. Added Met formed Met-S=O
with H
2
O
2
; therefore, added Met reduced or used up
H
2
O
2
. In this way, the inhibition of H
2
O
2
could be
prevented. As described in possible result 2, Met-S=O
isn't all turned back into Met. Thus, gradually
melanogenesis is declined. H
2
O
2
of low concentration
( 10-6 M ) activated tyrosinase increasing the
maximum reaction rates/5min for more than 2 fold
over control values in the absence of any H
2
O
2
. Rates
for tyrosinase/5min across the range of H
2
O
2
from 0
to 40×10-3 M increased from 49 μmol
tyrosinase/5min to 105 μmol tyrosinase/5min.
3.1 Possible Result 1
Experimental group (HFMs, Met, PC-KUS) shows a
strong expression of catalase, MSRA, and MSRA.
Reaction rate of tyrosinase/5min increases over 4-
fold.
Met added scavenged H
2
O
2
by forming Met-S=O;
and PC-KUS decreases concentration of H
2
O
2
,
leading to a low concentration of H
2
O
2
of 50×10-3 M.
Tyrosinase is activated.
ICBB 2022 - International Conference on Biotechnology and Biomedicine
94

3.2 Possible Result 2
Experimental group (HFMs, Met, PC-KUS) shows a
weaker expression of catalase, MSRA, and MSRA.
Reaction rate of tyrosinase/5min increases over 1.5-
fold.
Met or PC-KUS is excessive to completely react
with H
2
O
2
. H
2
O
2
is absent, making little difference to
reaction rate and activity of tyrosinase.
Table 1 Possible Results.
Group (HFMs, 10
-3
M H
2
O
2
) Result 1 Result 2
Group 1(+ Methionine) ++ ++
Grou
p
2
(
+
p
seudocatalase
)
++ ++
Grou
p
3
(
raw
)
--
Grou
p
4
(
+Methionine,
p
seudocatalase
)
+++ +
Note. Table 1 shows the two possible results of the
experiment. For result 1, the activation is obvious; for
result 2, the effect of combining is not as good as that
of separating. “+” represents an increase in tyrosinase
activity. “-” represents a decrease in tyrosinase
activity. “+-” represents not significantly different
from negative control. “?” represents uncertain
results.
4 DISCUSSION
This is the first research on the combined functioning
of H
2
O
2
and Met.
The result of group 1 can be attained from the
interpretation that free Methionine alters Methionine
on tyrosinase. There’s Methionine on tyrosinase.
H
2
O
2
oxidizes the Methionine and thus inhibits the
enzyme. However, if extra Methionine is added, it
will be added Methionine which is oxidized instead
of Methionine on tyrosinase.
The result of group 2 is obtained on the analogy
of PC-KUS’s treatment of vitiligo (Schallreuter,
2004). Based on the similarity in the pathology of
vitiligo and hair graying, pseudocatalase is supposed
to affect tyrosinase similarly as it does to
acetylcholinesterase (AchE), whose deactivation is
critical for vitiligo. Although it’s certain that a low
concentration of H
2
O
2
(10-6 M) activates AchE, the
mechanism is unknown.
In possible result 2, H
2
O
2
is excessive, making
PC-KUS and Methionine react completely. There
may be a slight decrease in H
2
O
2
concentration, but
the change is not enough for an obvious increase in
tyrosinase activity.
5 CONCLUSION
In summary, the research proves the activation of
H
2
O
2
and Met to tyrosinase, respectively, and it
investigates the validity of H
2
O
2
and Met to
tyrosinase activity in combination. My study’s
findings will indicate whether the activity of
tyrosinase activated by H
2
O
2
and Met reaches a
higher level than when they are used alone. My
research findings may provide a solution to the
problem of hair graying. It may be possible to remain
“perpetually young and vital” for a longer period.
Even though low concentrations of H
2
O
2
can
activate enzymes, the mechanism by which it works
is unknown. Experiments only demonstrate that
increasing the concentration of H
2
O
2
within a certain
range activates enzymes more and more effectively;
and that activation terminates when a certain amount
is exceeded. Additional experiments are required to
determine the precise ultimate quantity, which may
vary between enzymes. Additionally, while
pseudocatalase acts similarly to natural catalase in
that it degrades H
2
O
2
to water and oxygen, the effect
of PC-KUS treatment on hair graying requires further
investigation.
REFERENCES
Ikeda, K., Masujima, T., & Sugiyama, M. (1996). Effects
of Methionine and Cu2+ on the Expression of
Tyrosinase Activity in Streptomyces
castaneoglobisporus. Journal Of Biochemistry, 120(6),
1141-1145. doi:
10.1093/oxfordjournals.jbchem.a021533
Methionine - Health Encyclopedia - University of
Rochester Medical Center. (n.d.). University of
Rochester Medical Center.
https://www.urmc.rochester.edu/encyclopedia/content.
aspx?contenttypeid=19&contentid=Methionine
Sextius, P., Betts, R., Benkhalifa, I., Commo, S., Eilstein,
J., & Massironi, M. et al. (2017). Polygonum
multiflorum Radix extract protects human foreskin
melanocytes from oxidative stress in vitro and
potentiates hair follicle pigmentation ex vivo.
International Journal of Cosmetic Science, 39(4), 419-
425. doi: 10.1111/ics.12391
Synergy of H2O2 and Methionine Affects Tyrosinase Activity, Causing Repigmentation of Gray Hair
95

Schallreuter, K. U., Elwary, S. M., Gibbons, N. C., Rokos,
H., & Wood, J. M. (2004). Activation/deactivation of
acetylcholinesterase by H
2
O
2
: more evidence for
oxidative stress in vitiligo. Biochemical and
Biophysical Research Communications, 315(2), 502–
508. https://doi.org/10.1016/j.bbrc.2004.01.082
Wood, J., Decker, H., Hartmann, H., Chavan, B., Rokos,
H., & Spencer, J. et al. (2009). Senile hair graying: H 2
O 2 ‐mediated oxidative stress affects human hair color
by blunting Methionine sulfoxide repair. The FASEB
Journal, 23(7), 2065-2075. doi: 10.1096/fj.08-125435
ICBB 2022 - International Conference on Biotechnology and Biomedicine
96