8
1 Melanin Chemistry
presence of tyrosinase was also subsequently reported in some of the plants, the
insects, the fungi, and the marine organisms. Thus, the presence of mammalian
tyrosinase was also expected [28]. However, identification of mammalian tyrosinase
had been not straightforward to conclude for several years.
As an example, extracts of horse melanoma were able to convert tyrosine to
melanin, whereas no tyrosinase activity was demonstrated by using fetal rabbit skin
[29]. Bloch demonstrated important findings and tried to explain this puzzle, although
not completely successful. Bloch immersed frozen sections of pigmented human skin
in a solution of 3,4-dihydroxyphenylalanine (dopa), which is a hydroxylated tyrosine.
The immersed tissues presented black pigments. This method is used even today
to confirm the presence of melanocytes, and called the dopa staining. In contrast,
immersing in tyrosine did not result in this pigment formation. From this result, Bloch
proposed that “dopa-oxidase”, which catalyzes the oxidation of dopa, is present in
mammalian skins, whereas tyrosinase does not exist [30].
In this connection, it is noted that Raper isolated dopa in crystalline form, which
was generated by oxidizing tyrosine with plant- or insect-derived tyrosinase [28]. The
formed dopa could be further oxidized to form the final product melanin. Therefore,
dopa is an intermediate of melanogenesis of, at least, the plants and the insects.
In 1942, Hogeboom and Adams demonstrated the oxidation of tyrosine and dopa
when added to extracts of mouse melanoma, as confirmed by oxygen uptake. This
oxidation was followed by black pigment formation, and thus the presence of tyrosinase as well as dopa-oxidase in mammalian tumors was indicated [30]. The tyrosinase
and dopa-oxidase activity were separately obtained by fractionation using ammonium
sulfates solution upon centrifugation of the melanoma.
Greenstein et al. also found tyrosinase and dopa-oxidase activity in the extracts of
human melanoma [29]. The above studies on mammalian melanomas demonstrated
lower tyrosinase activity than that of dopa-oxidase in general, and then tyrosinase
activity could be hardly extractable depending on the tissues. Use of melanomas,
which arose from sufficient quantities of melanocytes, is thought to be advantageous
for finding the tyrosinase activity.
However, the hypothesis of co-existing tyrosinase and dopa-oxidase was denied
after a while. In 1949, Lerner et al. reinvestigated tyrosinase activity from mouse
melanoma [31]. The tyrosinase-catalyzed oxidation is initially very slow or lagged
before the reaction begins, and then becomes fast later. This lag is referred to as
the induction period. Lerner et al. found a shortened induction period by adding
dopa, and formation of dopa by this catalyzed oxidation as an intermediate. It was
also pointed out that dopa is readily oxidized even without enzymes above pH 7.0,
indicating that the previous study by Bloch could overestimate the activity on dopa
oxidation since the pH was at 7.4.
Lerner et al. emphasized the difficulties in separately evaluating the activity on
tyrosine and dopa oxidation upon fractionation in the presence of the factor reducing
the induction period. Based on this point, Lerner et al. proposed that tyrosinase and
the previously hypothesized dopa-oxidase must be considered as the same enzyme,
and thus the term tyrosinase should be only recommended.
1 Melanin Chemistry
presence of tyrosinase was also subsequently reported in some of the plants, the
insects, the fungi, and the marine organisms. Thus, the presence of mammalian
tyrosinase was also expected [28]. However, identification of mammalian tyrosinase
had been not straightforward to conclude for several years.
As an example, extracts of horse melanoma were able to convert tyrosine to
melanin, whereas no tyrosinase activity was demonstrated by using fetal rabbit skin
[29]. Bloch demonstrated important findings and tried to explain this puzzle, although
not completely successful. Bloch immersed frozen sections of pigmented human skin
in a solution of 3,4-dihydroxyphenylalanine (dopa), which is a hydroxylated tyrosine.
The immersed tissues presented black pigments. This method is used even today
to confirm the presence of melanocytes, and called the dopa staining. In contrast,
immersing in tyrosine did not result in this pigment formation. From this result, Bloch
proposed that “dopa-oxidase”, which catalyzes the oxidation of dopa, is present in
mammalian skins, whereas tyrosinase does not exist [30].
In this connection, it is noted that Raper isolated dopa in crystalline form, which
was generated by oxidizing tyrosine with plant- or insect-derived tyrosinase [28]. The
formed dopa could be further oxidized to form the final product melanin. Therefore,
dopa is an intermediate of melanogenesis of, at least, the plants and the insects.
In 1942, Hogeboom and Adams demonstrated the oxidation of tyrosine and dopa
when added to extracts of mouse melanoma, as confirmed by oxygen uptake. This
oxidation was followed by black pigment formation, and thus the presence of tyrosinase as well as dopa-oxidase in mammalian tumors was indicated [30]. The tyrosinase
and dopa-oxidase activity were separately obtained by fractionation using ammonium
sulfates solution upon centrifugation of the melanoma.
Greenstein et al. also found tyrosinase and dopa-oxidase activity in the extracts of
human melanoma [29]. The above studies on mammalian melanomas demonstrated
lower tyrosinase activity than that of dopa-oxidase in general, and then tyrosinase
activity could be hardly extractable depending on the tissues. Use of melanomas,
which arose from sufficient quantities of melanocytes, is thought to be advantageous
for finding the tyrosinase activity.
However, the hypothesis of co-existing tyrosinase and dopa-oxidase was denied
after a while. In 1949, Lerner et al. reinvestigated tyrosinase activity from mouse
melanoma [31]. The tyrosinase-catalyzed oxidation is initially very slow or lagged
before the reaction begins, and then becomes fast later. This lag is referred to as
the induction period. Lerner et al. found a shortened induction period by adding
dopa, and formation of dopa by this catalyzed oxidation as an intermediate. It was
also pointed out that dopa is readily oxidized even without enzymes above pH 7.0,
indicating that the previous study by Bloch could overestimate the activity on dopa
oxidation since the pH was at 7.4.
Lerner et al. emphasized the difficulties in separately evaluating the activity on
tyrosine and dopa oxidation upon fractionation in the presence of the factor reducing
the induction period. Based on this point, Lerner et al. proposed that tyrosinase and
the previously hypothesized dopa-oxidase must be considered as the same enzyme,
and thus the term tyrosinase should be only recommended.