Received for publication May 19, 1989
and in revised form June 30, 1989
Plant Physiol. (1989) 91, 481-483
0032-0889/89/91/0481 /03/$01 .00/0
Communication
Polypeptide Composition and Amino-Terminal Sequence of
Broad Bean Polyphenoloxidase'
William H. Flurkey
Department of Chemistry, Indiana State University, Terre Haute, Indiana 47809
differences in molecular size (5, 11). Within a given plant
species, however, some of these enzyme forms may be derived
from a common parent form. If so, other molecular events or
cellular processes must be responsible for generation of these
enzyme forms which differ in mol wt. In an attempt to
understand how these different enzyme forms in broad bean
PPO arise, we have purified the enzyme and examined its
polypeptide composition by SDS-PAGE (Table I). Individual
polypeptides from SDS-PAGE samples were sequenced at the
amino terminal end and analyzed for homology and structural
information. These results suggest that individual polypeptides which have different mol wt may be modified to account
for dissimilar size, but they contain a similar N-terminal
sequence. This is the first report detailing the composition
and amino-terminal sequence of the purified enzyme.
ABSTRACT
Polyphenoloxidase was purified from broad bean (Vicia faba)
leaves. The purified enzyme contained two immunocross-reactive
proteins of approximately 60 to 65 and 43 to 45 kilodaltons.
Further electrophoretic separation resolved these proteins into
doublets with molecular mass of 61.5, 60, 44.5, and 43 kilodaltons,
respectively. Each of the four polypeptides was transferred to
Immobilon and subjected to microprotein sequencing. All the
polypeptides showed the same amino acid sequence up to residue 9 but some variations occurred thereafter. The amino-terminal sequence contained a large number of proline and serine
residues. These results suggest that the four polypeptides were
derived from a common parent form and that a posttranslational
modification(s) must have occurred to account for the difference
in their apparent size.
MATERIALS AND METHODS
Purification of PPO
PPO2 is an enigmatic chloroplast enzyme whose function(s)
still remains to be elucidated (for reviews see refs. 14, 15, 19,
20). The enzyme is ubiquitous and is found in a variety of
multiple forms. Many of these forms, especially those found
in broad beans, can be activated from a latent state by SDS
(1, 5, 7, 10, 17). Treatment of PPO with trypsin has also been
used to activate the enzyme ( 18) and to show generation of a
slightly lower molecular mass form (about 43 kD) of a purified
broad bean enzyme which still retained catalytic activity (8).
Although the enzyme has been studied frequently with regard
to its enzymatic and electrophoretic properties, little is known
about the structure and composition of the purified enzyme.
Composition and structural analysis has been difficult to
obtain because of the limited amounts of purified enzyme
available. For these reasons the knowledge of the mechanism
of activation and how different isoenzyme forms arise is
Broad bean (Vicia faba) PPO was purified from isolated
leaf chloroplast membranes on a small scale as described by
Hutcheson et al. (6). The enzyme was isolated from acetone
powders of broad bean leaves on a larger scale. Briefly, acetone
powders (100 g) were suspended in 10 vol of 10 mm sodium
phosphate (7.0), stirred for 1 to 4 h, and centrifuged. The
supernatant was treated with 100 g of DEAE (equilibrated at
pH 7.0) cellulose batchwise to remove phenolics and was
filtered. The filtrate was treated with CM cellulose, also preequilibrated at pH 7.0, stirred, and filtered. The filtrate was
concentrated and passed through DEAE cellulose, Sephadex
G- 100, and hydroxylapatite columns as described by Hutcheson et al. (6). At this stage several preparations were combined.
Significant amounts of contaminating material were still present, and the combined extracts were passed through the
DEAE, Sephadex G- 100, and hydroxylapaptite columns until
no further contaminants were apparent on SDS-PAGE.
scarce.
Broad bean PPO has been suggested to be synthesized as a
45 kD protein which lacks a transit peptide needed for incorporation into the plant chloroplast (4, 5). Other plant PPOs
were also examined and found to be synthesized with a similar
mol wt as the broad bean enzyme (5). After partially denaturing and denaturing SDS-PAGE, many of these same plants
also showed active and inactive forms of PPO with apparent
Electrophoresis and Electrotransfer
Denaturing SDS-PAGE was carried out in 6.0 to 6.5%, 9%,
and 10 to 20% (30.8% T, 2.6% C) polyacrylamide gels using
the Laemmli system (9). Samples were boiled and made 2%
in SDS and 1% in mercaptoethanol before application. Electrophoresis was performed in 8 x 10 cm mini-gels or in larger
vertical 10 x 10 cm mini-gels. Proteins were transferred from
SDS gels to PVDF (Immobilon) membranes in 10 mM CAPS/
' Part of this research was supported from a grant from the Campbell Institute for Research and Technology (Napoleon, OH).
2
Abbreviation: PPO, polyphenoloxidase.
481
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Plant Physiol. Vol. 91,1989
FLURKEY
482
10% methanol buffer (pH 11) at room temperature as described by Matsudaira (13). The membranes were stained and
destained with Coomassie blue R-250 for protein sequencing
according to the manufacturer's instructions (Millipore
Corp.). Similar samples were also processed for Western blotting as described by Lanker et al. (10, 11) using either polyclonal or monoclonal antibodies to PPO.
1
2
95.5
~
~
Protein Sequencing
Four denatured polypeptides bound to Immobilon were
sequenced at the amino-terminal end using microprotein
sequencing techniques. Protein bands were cut out of the
Immobilon and centered on the Teflon seal and then placed
in the cartridge block. Proteins were sequenced using an
Applied Biosystems model 479 A Gas Phase Sequenator
equipped for on-line PTH analysis (12, 13). The PTH derivatives were separated and analyzed by reverse phase HPLC
on C18 columns (12, 13). Sequencing on Immobilon membranes was carried out by the Protein Sequencing Laboratory
at the Whitehead Institute for Biomedical Research at the
Massachusetts Institute of Technology.
RESULTS AND DISCUSSION
Broad bean PPO was previously purified by Hutcheson et
al. (6) to make antibodies to PPO and to determine if PPO
was involved in light-dependent photochemical oxidation of
polyphenols. Native electrophoresis showed the possible existence of more than one protein in their purified PPO preparation. Unfortunately, no data were provided on the purity
of their preparation by SDS-PAGE. Our purified preparations
consistently contained two polypeptides of approximately 60
to 65 kD and 43 to 45 kD in 9% SDS-PAGE (Fig. 1). Both
of these bands appeared to be diffuse and lacked the tightness
in band width associated with many proteins after SDSPAGE. Both of these polypeptides were also found in crude
cellular extracts of broad bean leaves and were cross-reactive
to polyclonal and monoclonal antibodies against broad bean
PPO (10, 1 1). During the course of many purifications, I have
noticed that the ratio of these two polypeptides varied from
preparation to preparation but we have not been able to
ascertain the cause of this variability (4, 8; data not shown).
In addition, these two polypeptides were not separable by ion
exchange, gel filtration, hydrophobic, or immunoaffinity
chromatography. Because the polypeptides appeared diffuse,
and thus suggested possible heterogeneity, further electrophoretic experiments were conducted.
Table I. Amino-Terminal Sequence of Polypeptides of Broad Bean
PPO
Amino Acid Residue
Polypeptide
2 3 4 5
P I S P
P I S P
P I S P
Bi
P I S P
B2
aDash represents a residue
identified.
Al
A2
1
S
S
S
S
6 7
P D
P D
P D
P D
which
8 9 10 11 12 13 14
L S K -a G P P
L S - I G P P
L S K - G
L S K N G
could not be determined or
4
f
~
~
~
5
6.
8
M
.,.
.
~
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Figure 1. SDS-PAGE analysis of purified broad bean PPO polypeptides in different electrophoretic conditions. Lane 1, purified PPO run
on a 9% gel; lane 2, mol wt markers; lane 3, purified PPO run on a
6.3% gel and run to the lower third of the gel; lane 4, 7 polypeptides
(Al, A2, B1, B2) cut out from a duplicate gel of lane 3 and rerun on
a 7.5% gel; lane 8, 55 and 36 kD molecular mass markers. Al, A2,
B1, and B2 designate polypeptides found in the purified PPO.
When purified PPO was subjected to gel concentrations
ranging from 6.0 to 6.5%, SDS-PAGE analysis showed that
each of the broad bands at 60 to 65 and 43 to 45 kD was split
into a doublet (Fig. 1). None of the resulting polypeptides
were particularly sharp and the doublets tended to diffuse
together. By electrophoresing the bands in the lower third of
the gel, the doublets could be separated by a few millimeters.
In spite of the diffuse nature of the bands, they were easily
distinguishable and an approximate Mr could be obtained.
Two polypeptides, designated as Al and A2, were characterized with molecular mass of 61.5 and 60 kD. The ratio of the
protein staining intensity in these two proteins was approximately 2:1. Two other polypeptides, designated B 1 and B2,
were also observed in the 43 to 45 kD region and were
characterized with molecular mass of 44.5 and 43 kD. Each
of these bands showed equal protein staining intensity. To
show that each of these four proteins was distinct, they were
excised from the gel and rerun on another SDS-PAGE (Fig.
1). Each protein migrated at a characteristic position and mol
wt, but the banding patterns were again diffuse. Each of these
proteins also cross-reacted with antibodies to PPO after Western blotting (data not shown). Taken together, these results
indicated that the purified broad bean PPO contained several
proteins of dissimilar mol wt and even closely related mol wt
species may be composed of heterogeneous population.
To determine if these polypeptides were derived from a
common parent form, the purified PPO was subjected to
SDS-PAGE to separate the four polypeptides. They were
transferred to Immobilon and each of the four proteins was
subjected to amino terminal micro-protein sequencing. Areas
from each protein band were cut out to minimize crosscontamination with closely related migrating species. Even
so, several problems were encountered in the attempts to
sequence these proteins. Difficulties were encountered in scaling up the electrophoresis to obtain sufficient mass for se-
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COMPOSITION AND N-TERMINAL SEQUENCE OF POLYPHENOLOXIDASE
quencing while still retaining the capability to separate the
proteins adequately. Greater mass amounts of these proteins
were required for sequencing because initial sequencing experiments indicated a large number of proline residues in the
N-terminal sequence. The presence of proline residues resulted in decreased yields during the subsequent sequencing
steps. Finally, during transfer onto Immobilon, the bands
tended to diffuse. These difficulties were overcome and the
amino terminal sequences were successfully determined for
Al and A2 up to 14 amino acids. Proteins B 1 and B2 were
sequenced up through 10 amino acids.
Since the sequence of each polypeptide was determined,
none of these proteins contained a blocked amino terminus.
From the sequence analysis, it is readily apparent that each
of the four proteins has identical N-terminal sequences up to
residue 9. Residue 10 could not be determined with a degree
of certainty in A2. At residue 11 there was some difficulty in
assigning amino acids in Al and B 1, but A2 appeared to
contain a different amino acid from B2. Even in this short
sequence, a large number of prolines and serines were present.
Computer analysis of the short sequence in A 1, using the
Beckman Microgenie program for protein homology and
structural analysis, indicated that there was no homology to
any proteins in the data bank. Predictions for a-helix were
negative. Some potential existed for fl-sheets and a positive
prediction was made for random coils and positive
hydrophilicity.
I have previously shown (4, 5, 10) that the primary translation product for broad bean PPO was approximately 45 kD
and was found in several stages of leaf development. Presumably, this product corresponds to one of the proteins found in
the 43 to 45 kD molecular mass region in the isolated protein.
If so, then one of these two proteins has been modified from
the original translation product to account for the small
decrease in observed mol wt. Because all four proteins have a
similar amino terminal sequence, substantial modification(s)
must also have occurred in Al and A2 in order to account
for the large increase in apparent mol wt. The type of modification(s) which appears in these two proteins has not been
determined and awaits further characterization. These modifications may include glycosylation since Robb et al. (16) and
Biehl et al. (3) have reported carbohydrate associated with
broad bean and spinach PPO, respectively. These higher mol
wt proteins may also contain covalently bound phenolic
groups because some PPO are known to be tanned (2). Further
sequence analysis will be valuable in elucidating specific
amino acid modifications or attachments in these four proteins and also be useful for generating specific oligonucleotide
probes for cloning and gene analysis.
ACKNOWLEDGMENTS
We thank Bob Buchahan for the use of his polyclonal antibody to
broad bean PPO. We also thank Paul Matsudaira and Susan Black-
483
man at the Protein Sequencing Laboratory at the Whitehead Institute
for Biomedical Research at the Massachusetts Institute of Technology
for the protein sequencing experiments. We appreciate comments by
S. Duke, K. Vaughn, and A. Lax. Secretarial assistance was provided
by P. Archer and K. Divan.
LITERATURE CITED
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plant and fungal polyphenoloxidases isoenzymes in sodium
dodecyl sulfate electrophoresis. Phytochemistry 23: 2723-2725
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In JB Pridham, ed, Enzyme Chemistry of Phenolic Compounds. Pergamon Press, Oxford, pp 7-24
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phenolase and membrane integrity. International Symposium
on Membranes and Compartmentation in Regulation of Plant
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5. Flurkey WH (1986) Polyphenoloxidase in higher plants: immunological detection and analysis of in vitro translation products. Plant Physiol 81: 614-618
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the tyrosinase of broad bean ( Vicia faba L.) Phytochemistry 4:
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(L.) tyrosinase as effected by denaturing agents. Phytochemistry 5: 469-482
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