Vol. 69, No. 3,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE COMPLETE AMINO ACID SEQUENCE OF THE SPIRULINA Masaru Department
Tanaka,
Misturu
Haniu
of Biochemistry-Biophysics,
and Kerry
of Hawaii
Hawaii
FERREDOXIN
T. Yasunobu*
University Honolulu,
PLATENSIS
School
of Medicine
96822
and Krishna Botany
Department,
University
Received
February
3,1976
K. Rao and David of London
0. Hall
King's
College,
London,
England
The complete amino acid sequence of ferredoxin from the Spirulina a blue green non-filamentous algae has been determined. The amino acid sequence differs from the reported sequence of Wada --' et al (1) at residue 9. Our results indicate that the residue is Asn while Wada it to be Asp. In addition, residues 86 and 87 have --et al. have reported been determined in our study while Wada --et al. have assumed these residues by homology to other ferredoxins which have been sequenced. Modifications of the automated sequence procedure previously described (2) were again found to be useful for peptide sequencing. e:S
Eucaryotic
ferredoxins
to date
(3)
include
alfalfa
(6),
taro
of the plant
the proteins (7),
has been completely
ferredoxin
(9).
Nearly
another
by Wada --et al. ently
just
ferredoxin results
the complete
the amino
by an independent from
EXPERIMENTAL Spirulina
the
sequence
two laboratories
Leucaena
glauca
the Spirulina
(5),
maxima
of the Spirulina
platensis
been reported
Our laboratory
has independ-
of the -S. platensis
from Wada --et al.
are compared
sequenced
one procaryotic
has just
sequence
procedure
have been
(4),
namely
note.
acid
which
Only
ferredoxin
in a preliminary
completed
(8).
sequenced,
procaryotic
(1)
spinach
and Scenedesmus
ferredoxin
Eerredoxin,
from
type
in this
(1)
and the sequence
report,
PROCEDURES platensis
ferredoxin.
Ferredoxin was extracted and purified from the dried Spirulina platensis cells by the procedure described previously for the Spirulina maxima (10). The preparation of the carboxymethylferredoxin has been described previously (9). Trypsin digestion filtration -of carboxymethylferredoxin -and & ___--of
t: To whom requests
for
reprints
Copyrighf 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
should 759
be addressed.
BIOCHEMICAL
Vol. 69, No. 3, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
About 25 mg of carboxymethylferredoxin were digested tryptic digest. with 4% TPCK-trypsin (11) in pH 8 at 28' for 24 hours. The dried digest was applied to a Sephadex G-50 column (1.9 x 57 cm). The solvent used was 0.1 N ammonium hvdroxide. Isoelectric focusing of the mixture of peptides, T2 and T3 and ---F further purification The lyophilized m=ure --of thFixa=tides. (5of peptide T2 and T3 obtained from the first peak during gel filtration of the trpptic digest of carboxymethylferredoxFn was separated into two peptides, T2 and T3, by electrofocusing techniques The applied voltage was (12) using ampholine (pH range of 3.5 - 10). 30C volts and a column of 148 ml capacity was used. After 11.5 hours, prior to the attainment of an equilibrium state, fractions of 3.5 ml were Each peak collected and the pH of the various samples were determined. fraction was again rechromatographed on a Sephadex G-50 column. Sequence determinations and amino acid analyses. Amino-terminal end group analyses of the proteinGdxiK(l55 to 300 nmoles) were carried out by the manual Edman degradation (13) or by the automated Edman degradation procedure (14) in the Beckman-Spinco Model 890 Protein/Peptide Sequencer. All of the automated runs utilized the protein double cleavage program and 0.2 M Quadrol as the coupling buffer. Prior to the Sequencer analysis, peptide T3 was reacted with 4-sulfophenylisothiocyanate (15). The phenylthiohydantoins of the amino acids were determined by gas chromatography (16) or by thin layer chromatography (14), or by amino acid analyses of the 6N HCl hydrolysates of the amino acid phenylthiohydantoins (17). COOH-terminal end groups of the protein and peptides were determined by the action of carboxypeptidase B and/ or A digestion (18), and/ or by hydrazinolysis experiments (19). The amino acid compositions of the protein and peptides were determined on 6N HCl hydrolysates in a Beckman-Spinco Model 12OC automatic amino acid analyzer (20). RESULTS --Amino acid ferredoxin. was determined ferredoxin terminal
composition
The amino
acid
composition
on 24-,
48-,
and 72-hours
and the protein amino
(100% yield)
acid
carboxymethylferredoxin digestion
(6 hours)
ferredoxin
yielded
analyses of the
S. platensis
Sequencer yielded
followed glycine
hydrolysates
Thus,
the COOH-terminal
shown
to be -Gly-Leu-Tyr-COOH.
analysis,
sequence
The NH2-
was shown to be alanine
in 86% yield.
of the Carboxypeptidase
of the carboxymethyl(loo%),
The end group
‘760
ferredoxin
of carboxymethyl-
and tyrosine
of the 5. platensis
was pure.
platensis
Hydrazinolysis
by hydrazinolysis leucine
platensis
of 98 residues.
ferredoxin
tyrosine
(79%),
-of Spirulina
Spirulina
was shown to consist
of the
by Protein
the ferredoxin
and end group --
analyses
ferredoxin indicated
(100%). was that
A
Vol. 69, No. 3, 1976
BIOCHEMICAL
Separation
&purification
of tryptic
of carboxymethylferredoxin chromatography. long
The second
fractions,
their
in Table
Sequence nmole)
amino
for
peaks the
G-50
The first
a small
was used
contained
further
contained
two
of undegraded
to separate
peptides
purification
compositions
peptides
column
peak
amount
peptides
T4 and Tl, of the peptide
and properties
analysis
of carboxymethylferredoxin
of the
was performed
Spirulina
twice
are
summarized
of Pth-alanine'
(Residues
platensis
the first
In a typical
experiment,
(300
Protein/Peptide
45 residues
was 94% and the recovery
l-45),
carboxymethylferredoxin
in the Beckman-Spinco
to determine
end of the protein.
from the
the average
of Pth-alanine
Sequencer. amino-terminal
repetitive at the
yield
forty-fifth
was 7%. Sequencer
43-90). both
analyses
and COOH-terminal --
The sequence the
analyses
peptide
4-sulfophenylthiocarbamyl
24 was removed performed
manually.
an additional leucine
from
the
cup.
By this
23 residues
The COOH-terminal B procedure
of experiment,
The abbreviation
derivative (300 nmole).
Subsequent
(2),
used
is:
Pth,
(300
it
nmole)
run of
peptide
after
steps
was possible
The recovery
of the peptide
6 hours
of the peptide
on
step
were to sequence
of Pth-iso-
was 10%.
and the carboxypeptidase after
out
Edman degradation
of the peptide.
analyses
-T3 (Residues
T3 was carried
the residual
modification
step
of peptide
In the Sequencer
derivative,
at the forty-seventh
peptidase
analysis
of the peptide
4-sulfophenylthiocarbamyl
and the underivatized
1
observed.
technique
acid
--results
was possible
type
by Sephadex
with
and third
Tryptic
I.
Sequencer
the
were
together
The method
peptides.
separated
The isoelectrofocusing
respectively.
step
peaks
T2 and T3,
T2 and T3.
It
were
Three
peptides,
protein.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of digestion
T3 utilized B-A
with
phenylthiohydantoin.
761
procedure.
the carboxyIn the
carboxypeptidase
latter A,
Vol. 69, No. 3, 1976
TABLE I:
BIOCHEMICAL
Amino Acid
Compositiona
and Properties
Carboxymethyl-ferredoxin Amino
Acid
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Tryptic
Peptides
of the
from 2. platensis.
Tl
T2
T4
T3
Total
Residues
Carboxymethylcyetine
1.88
(2)
3.86
(4)
6
Aspartic
8.05
(81
6.07
(61
14
2.97
(3)
6.89 (7)
0.88
(1)
4.76
(5)
acid
Threonine
0.93
(1)
Serine Glutamic
acid
0.95
(1)
12 6
2.98
(3)
12 2
0.99
(1)
7 10
4.97
(5)
4.03
(4)
Proline
0.87
(1)
0.94
(1)
Glycine
1.99 4.00
(2) (4)
3.97
(4)
5.00
(5)
Valine
1.09
(1)
1.98 (21
Isoleucine
3.96
(4)
3.86
(4)
Leucine
3.95
(4)
2.02
(2)
1.00
(1)
1.98
(2)
2.01
(2)
0.95
(11
0.98
(11
1
0.95
(1)
2
Alanine
1.00
Tyrosine
(1)
0.97
(1)
Phenylalanine Lysine
0.98
(1)
Histidine
0.91
Arginine Total
0.87 residues
Recovery Color
3 8
(%)
reaction
with
4 78 Purple
1
(1)
1
(1) 38
48
8
77 Purple
77 Purple
84
98
Yellow to Purple
ninhydrin Purification
7 6
methodb
PIN Rf= 0.67
Electro-
Electro-
focusing
focusing
BPAW Rf= 0.49
a
Results from 6N HCl hydrolyzates refer to the assumed stoichiometric peptide.
‘b
The abbreviations used are: PIN, paper chromatography in the solvent system, pyridine/isosmyl alcohol/O.1 N ammonium hydroxide (60 : 30 : 50, v/v); and BPAW, paper chromatography with 1-butanol/pyridine/acetic acid/water (60 : 40 : 12 : 48, v/v).
yield
in addition
The sequence Sequence
data
(24 and 48 hours). The numbers in parentheses number of residues per molecule of pure
to threonine of peptide
determination
(54%), T3 are
isoleucine summarized
-of peptide
Edman degradation
established
(200
After
step
the
seventh
in Figure
T4 (Residues
of the manual nmole).
(loo%),
91-98).
the sequence
of Edman degradation, 762
and lysine
(100%).
1. Seven steps of this free
peptide tyrosine
Vol.69,No.3,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1 5 10 2N-A~a-Thr-Tyr-Lys-Val-Thr-Leu-Ile-Asn-Glu-Ala-Glu-Gly-Ile-Asn-Glu-Thr-Ile-Asp-Cys + -+ + + + + -f + -+ -+ + -f -+ * Tl
15
20
-+
*
-+
+
+
+
T2
I
21 25 30 Asp-Asp-Asp-Thr-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-f -+ + -f -f -+ + -t + -+ + -f -+ -+
35. +
40 -f
-+
-+
-t
+
T2
C~~-Arg-Ala-Gly-A~~-Cys-Ser-Thr-Cys-A~~-Gly-Thr-Ile-Thr-S~~-Gly-Thr-Il~-Asp-G~~+ + + -+ -+
T3
T4
---7----Y
7--r--/-
-'c--r
Figure 1: Reconstructrion of the complete amino platensis ferredoxin and sequence data of peptide figure, the symbols ---) s-3-, and D determined by use of the Beckman-Spine0 Model 890 direct manual Edman degradation, carboxypeptidase hydrazinolysis experiments, respectively,
hydrazinolysis was quantitatively and the recovery Figure
of the digest determined of tyrosine
yielded in the was 49%.
l
acid sequence of Spirulina fragments. In the represent sequences ProteinfPeptide Sequencer, B and/or A digestion, and
S-B-carboxymethylcysteine sample
by direct
The results
1.
763
amino obtained
in 27% acid
analysis
are
summarized
in
Vol. 69, No. 3, 1976
Complete platensis
BIOCHEMICAL
sequence.
The Protein
ferredoxin
and that
it
order.
showed
was followed
Peptide
T4 is
of the protein.
complete
amino
acid
that
Sequencer
peptide
by peptide the
analysis
AND BIOPHYSKAL
sequence
the
T2 and then
Spirulina
by peptide
peptide
sequence
are
run of the
Tl was the NH2-terminal
COOH-terminal All
RESEARCH COMMUNICATIONS
data
summarized
peptide
T3,
in this
from
the carboxyl-terminal
used
to establish
in Figure
the
1.
DISCUSSION Sequence
investigations
laboratories ary
with
data
since
of the
the goal
these
of obtaining
Fe-S proteins
the case of the -S. platensis the amino ently
acid
sequence
carried
out
the groups
arrived
difference
noted
acid
residue
analysis --et al. these
did
not
residues
assumption of the
In a previous automated
sequence
used in
the sequence
peptide
2
(1) and our
Our sequence
9 clearly
Asp.
showed
that
the sequence
constant
in
it
is Asn.
by our
for
and both there
showed
layer
of residues
the various
laboratory,
study
was one
that
amino
chromatographic In addition,
Wada
86 and 87 but
algal-plant complete
In
and independ-
However,
Our thin
relics.
approaches
simultaneously
the sequence.
was shown to be correct
S. platensis
was found
were
in several
and evolution-
to be ancient
same sequence.
determine were
thought
the
than
progress
in
two different
Wada --et al.
9 is Asn rather
of residue
are
determination
at nearly
are
structure-function
ferredoxin,
by both
in
ferredoxins
assumed
ferredoxins. sequence
This
investigations
ferredoxin2. report
from our
determination
(Z),
was described.
determination
to be a suitable
laboratory
of the
modification
for
This
a modification technique
S. platensis the sequence
of the again
ferredoxin
was
and
determination
T3.
The studies meeting in corrected is now in published experimental
reported here were completed in September 1975. At a Dec. 1975, Matsubara indicated that residue 9 has been to Asn. Thus, the sequence determination by the two groups complete agreement. However, it is important to have verification of the sequence, especially when different approaches have been used.
764
of
Vol. 69, No. 3, 1976
The amino siderable were
acid
homology
noted
sequence with
ferredoxins laboratory
AND BIOPHYSICAL
of the -S. platensis
the 2. maxima
in positions
and algal from our
BIOCHEMICAL
9, 52,
ferredoxin.
57, and 90.
have been compared (9)
and need not
RESEARCH COMMUNICATIONS
ferredoxin Only
four
The sequences
and discussed
be discussed
shows condifferences
of the other
in a previous
piant report
here.
ACKNOWLEDGEMENTS Research was supported in part by Grant GM 16784 and GM 16228 the National Institutes of Health, Grant GB 43448 from the National Science Foundation and the U.K. Science Research Council.
frvm
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 3. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Wada, K., Hase, T., Tokunaga, H., and Matsubara, H. (1975), .-FEBS Letters 55, 102. Tanaka, M., Haniu, M., Yasunobu, K.T., Rao, K.K., and Hall, D.O. (1975), Biochemistry 14, 5535. in Iron-Sulfur Proteins Yasunobu, K.T., and Tanaka, M., (1973), (Lovenberg, W., ed), Vol. II, pp. 27-130, Academic Press, New York. Matsubara, H., and Sasaki, R.M. (1968). J. --Biol. Chem. 243, 1732. Benson, A.M., and Yasunobu, K.T. (1969),-L. Chem. 244, 955. --Biol. Keresztes-Nagy, S., Perini, F., and Margoliash, E. (1969), J. Biol. Chem. 244, 981. Rao, K.K., and Matsubara, H. (197G), Biochem. --Biow. --Res. -Commun. 38, 500. Sugeno, K., and Matsubara, H. (1969), 2. -Biol. 2979. --Chem. s, Tanaka, M., Haniu, M., Zeitlin, S., Yasunobu, K.T., Evans, M.C.W., Rao, K.K., and Hall, D.O. (1975), Biochem. Biophys. 64 -Res. --Commun. --.I 399. Hall, D.O., Rao, K.K., and Cammack, R. (1972), Biochem. Biophys. --P.es. Commun. 5, 798. Wang, S.-S., and Carpenter, F.H. (1965), 2. --Biol. Chem. 3, 1619. Vesterberg, 0. (1971), Methods Enzymol. 2, 389. Edman, P., (1970), in Protein Sequence Determination (Needleman, S.B., ed), p. 211, Springer-Verlag Berlin, He-g, and New York. Edman, P., and Begg, G. (1967). Eur. J. Biochem. I, 80. --Braunitzer, G., Schrank, B., and Ruhfus, A. (lq70), Hoppe-Seyler's -Z. Physiol. Chem. 351, 1589. Pisano, J.J., and Bronzert, T.J. (1969), J. Biol. s. 235, 5597. Van Orden, H.O., and Carpenter, F.H. (1964), -Biochem. -Bio&. -Res. Commun. 14, 399. Ambler, R.B. (1967), Methods Enzymol. 11, 436. Bradbury, J.H. (1958)xem. J. 68, 475. Spackman, D.H., Moore, r and Stein, W.H. (1958), Anal. Chem. 30, 1190.
765
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE COMPLETE AMINO ACID SEQUENCE OF THE SPIRULINA Masaru Department
Tanaka,
Misturu
Haniu
of Biochemistry-Biophysics,
and Kerry
of Hawaii
Hawaii
FERREDOXIN
T. Yasunobu*
University Honolulu,
PLATENSIS
School
of Medicine
96822
and Krishna Botany
Department,
University
Received
February
3,1976
K. Rao and David of London
0. Hall
King's
College,
London,
England
The complete amino acid sequence of ferredoxin from the Spirulina a blue green non-filamentous algae has been determined. The amino acid sequence differs from the reported sequence of Wada --' et al (1) at residue 9. Our results indicate that the residue is Asn while Wada it to be Asp. In addition, residues 86 and 87 have --et al. have reported been determined in our study while Wada --et al. have assumed these residues by homology to other ferredoxins which have been sequenced. Modifications of the automated sequence procedure previously described (2) were again found to be useful for peptide sequencing. e:S
Eucaryotic
ferredoxins
to date
(3)
include
alfalfa
(6),
taro
of the plant
the proteins (7),
has been completely
ferredoxin
(9).
Nearly
another
by Wada --et al. ently
just
ferredoxin results
the complete
the amino
by an independent from
EXPERIMENTAL Spirulina
the
sequence
two laboratories
Leucaena
glauca
the Spirulina
(5),
maxima
of the Spirulina
platensis
been reported
Our laboratory
has independ-
of the -S. platensis
from Wada --et al.
are compared
sequenced
one procaryotic
has just
sequence
procedure
have been
(4),
namely
note.
acid
which
Only
ferredoxin
in a preliminary
completed
(8).
sequenced,
procaryotic
(1)
spinach
and Scenedesmus
ferredoxin
Eerredoxin,
from
type
in this
(1)
and the sequence
report,
PROCEDURES platensis
ferredoxin.
Ferredoxin was extracted and purified from the dried Spirulina platensis cells by the procedure described previously for the Spirulina maxima (10). The preparation of the carboxymethylferredoxin has been described previously (9). Trypsin digestion filtration -of carboxymethylferredoxin -and & ___--of
t: To whom requests
for
reprints
Copyrighf 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
should 759
be addressed.
BIOCHEMICAL
Vol. 69, No. 3, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
About 25 mg of carboxymethylferredoxin were digested tryptic digest. with 4% TPCK-trypsin (11) in pH 8 at 28' for 24 hours. The dried digest was applied to a Sephadex G-50 column (1.9 x 57 cm). The solvent used was 0.1 N ammonium hvdroxide. Isoelectric focusing of the mixture of peptides, T2 and T3 and ---F further purification The lyophilized m=ure --of thFixa=tides. (5of peptide T2 and T3 obtained from the first peak during gel filtration of the trpptic digest of carboxymethylferredoxFn was separated into two peptides, T2 and T3, by electrofocusing techniques The applied voltage was (12) using ampholine (pH range of 3.5 - 10). 30C volts and a column of 148 ml capacity was used. After 11.5 hours, prior to the attainment of an equilibrium state, fractions of 3.5 ml were Each peak collected and the pH of the various samples were determined. fraction was again rechromatographed on a Sephadex G-50 column. Sequence determinations and amino acid analyses. Amino-terminal end group analyses of the proteinGdxiK(l55 to 300 nmoles) were carried out by the manual Edman degradation (13) or by the automated Edman degradation procedure (14) in the Beckman-Spinco Model 890 Protein/Peptide Sequencer. All of the automated runs utilized the protein double cleavage program and 0.2 M Quadrol as the coupling buffer. Prior to the Sequencer analysis, peptide T3 was reacted with 4-sulfophenylisothiocyanate (15). The phenylthiohydantoins of the amino acids were determined by gas chromatography (16) or by thin layer chromatography (14), or by amino acid analyses of the 6N HCl hydrolysates of the amino acid phenylthiohydantoins (17). COOH-terminal end groups of the protein and peptides were determined by the action of carboxypeptidase B and/ or A digestion (18), and/ or by hydrazinolysis experiments (19). The amino acid compositions of the protein and peptides were determined on 6N HCl hydrolysates in a Beckman-Spinco Model 12OC automatic amino acid analyzer (20). RESULTS --Amino acid ferredoxin. was determined ferredoxin terminal
composition
The amino
acid
composition
on 24-,
48-,
and 72-hours
and the protein amino
(100% yield)
acid
carboxymethylferredoxin digestion
(6 hours)
ferredoxin
yielded
analyses of the
S. platensis
Sequencer yielded
followed glycine
hydrolysates
Thus,
the COOH-terminal
shown
to be -Gly-Leu-Tyr-COOH.
analysis,
sequence
The NH2-
was shown to be alanine
in 86% yield.
of the Carboxypeptidase
of the carboxymethyl(loo%),
The end group
‘760
ferredoxin
of carboxymethyl-
and tyrosine
of the 5. platensis
was pure.
platensis
Hydrazinolysis
by hydrazinolysis leucine
platensis
of 98 residues.
ferredoxin
tyrosine
(79%),
-of Spirulina
Spirulina
was shown to consist
of the
by Protein
the ferredoxin
and end group --
analyses
ferredoxin indicated
(100%). was that
A
Vol. 69, No. 3, 1976
BIOCHEMICAL
Separation
&purification
of tryptic
of carboxymethylferredoxin chromatography. long
The second
fractions,
their
in Table
Sequence nmole)
amino
for
peaks the
G-50
The first
a small
was used
contained
further
contained
two
of undegraded
to separate
peptides
purification
compositions
peptides
column
peak
amount
peptides
T4 and Tl, of the peptide
and properties
analysis
of carboxymethylferredoxin
of the
was performed
Spirulina
twice
are
summarized
of Pth-alanine'
(Residues
platensis
the first
In a typical
experiment,
(300
Protein/Peptide
45 residues
was 94% and the recovery
l-45),
carboxymethylferredoxin
in the Beckman-Spinco
to determine
end of the protein.
from the
the average
of Pth-alanine
Sequencer. amino-terminal
repetitive at the
yield
forty-fifth
was 7%. Sequencer
43-90). both
analyses
and COOH-terminal --
The sequence the
analyses
peptide
4-sulfophenylthiocarbamyl
24 was removed performed
manually.
an additional leucine
from
the
cup.
By this
23 residues
The COOH-terminal B procedure
of experiment,
The abbreviation
derivative (300 nmole).
Subsequent
(2),
used
is:
Pth,
(300
it
nmole)
run of
peptide
after
steps
was possible
The recovery
of the peptide
6 hours
of the peptide
on
step
were to sequence
of Pth-iso-
was 10%.
and the carboxypeptidase after
out
Edman degradation
of the peptide.
analyses
-T3 (Residues
T3 was carried
the residual
modification
step
of peptide
In the Sequencer
derivative,
at the forty-seventh
peptidase
analysis
of the peptide
4-sulfophenylthiocarbamyl
and the underivatized
1
observed.
technique
acid
--results
was possible
type
by Sephadex
with
and third
Tryptic
I.
Sequencer
the
were
together
The method
peptides.
separated
The isoelectrofocusing
respectively.
step
peaks
T2 and T3,
T2 and T3.
It
were
Three
peptides,
protein.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of digestion
T3 utilized B-A
with
phenylthiohydantoin.
761
procedure.
the carboxyIn the
carboxypeptidase
latter A,
Vol. 69, No. 3, 1976
TABLE I:
BIOCHEMICAL
Amino Acid
Compositiona
and Properties
Carboxymethyl-ferredoxin Amino
Acid
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Tryptic
Peptides
of the
from 2. platensis.
Tl
T2
T4
T3
Total
Residues
Carboxymethylcyetine
1.88
(2)
3.86
(4)
6
Aspartic
8.05
(81
6.07
(61
14
2.97
(3)
6.89 (7)
0.88
(1)
4.76
(5)
acid
Threonine
0.93
(1)
Serine Glutamic
acid
0.95
(1)
12 6
2.98
(3)
12 2
0.99
(1)
7 10
4.97
(5)
4.03
(4)
Proline
0.87
(1)
0.94
(1)
Glycine
1.99 4.00
(2) (4)
3.97
(4)
5.00
(5)
Valine
1.09
(1)
1.98 (21
Isoleucine
3.96
(4)
3.86
(4)
Leucine
3.95
(4)
2.02
(2)
1.00
(1)
1.98
(2)
2.01
(2)
0.95
(11
0.98
(11
1
0.95
(1)
2
Alanine
1.00
Tyrosine
(1)
0.97
(1)
Phenylalanine Lysine
0.98
(1)
Histidine
0.91
Arginine Total
0.87 residues
Recovery Color
3 8
(%)
reaction
with
4 78 Purple
1
(1)
1
(1) 38
48
8
77 Purple
77 Purple
84
98
Yellow to Purple
ninhydrin Purification
7 6
methodb
PIN Rf= 0.67
Electro-
Electro-
focusing
focusing
BPAW Rf= 0.49
a
Results from 6N HCl hydrolyzates refer to the assumed stoichiometric peptide.
‘b
The abbreviations used are: PIN, paper chromatography in the solvent system, pyridine/isosmyl alcohol/O.1 N ammonium hydroxide (60 : 30 : 50, v/v); and BPAW, paper chromatography with 1-butanol/pyridine/acetic acid/water (60 : 40 : 12 : 48, v/v).
yield
in addition
The sequence Sequence
data
(24 and 48 hours). The numbers in parentheses number of residues per molecule of pure
to threonine of peptide
determination
(54%), T3 are
isoleucine summarized
-of peptide
Edman degradation
established
(200
After
step
the
seventh
in Figure
T4 (Residues
of the manual nmole).
(loo%),
91-98).
the sequence
of Edman degradation, 762
and lysine
(100%).
1. Seven steps of this free
peptide tyrosine
Vol.69,No.3,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1 5 10 2N-A~a-Thr-Tyr-Lys-Val-Thr-Leu-Ile-Asn-Glu-Ala-Glu-Gly-Ile-Asn-Glu-Thr-Ile-Asp-Cys + -+ + + + + -f + -+ -+ + -f -+ * Tl
15
20
-+
*
-+
+
+
+
T2
I
21 25 30 Asp-Asp-Asp-Thr-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-f -+ + -f -f -+ + -t + -+ + -f -+ -+
35. +
40 -f
-+
-+
-t
+
T2
C~~-Arg-Ala-Gly-A~~-Cys-Ser-Thr-Cys-A~~-Gly-Thr-Ile-Thr-S~~-Gly-Thr-Il~-Asp-G~~+ + + -+ -+
T3
T4
---7----Y
7--r--/-
-'c--r
Figure 1: Reconstructrion of the complete amino platensis ferredoxin and sequence data of peptide figure, the symbols ---) s-3-, and D determined by use of the Beckman-Spine0 Model 890 direct manual Edman degradation, carboxypeptidase hydrazinolysis experiments, respectively,
hydrazinolysis was quantitatively and the recovery Figure
of the digest determined of tyrosine
yielded in the was 49%.
l
acid sequence of Spirulina fragments. In the represent sequences ProteinfPeptide Sequencer, B and/or A digestion, and
S-B-carboxymethylcysteine sample
by direct
The results
1.
763
amino obtained
in 27% acid
analysis
are
summarized
in
Vol. 69, No. 3, 1976
Complete platensis
BIOCHEMICAL
sequence.
The Protein
ferredoxin
and that
it
order.
showed
was followed
Peptide
T4 is
of the protein.
complete
amino
acid
that
Sequencer
peptide
by peptide the
analysis
AND BIOPHYSKAL
sequence
the
T2 and then
Spirulina
by peptide
peptide
sequence
are
run of the
Tl was the NH2-terminal
COOH-terminal All
RESEARCH COMMUNICATIONS
data
summarized
peptide
T3,
in this
from
the carboxyl-terminal
used
to establish
in Figure
the
1.
DISCUSSION Sequence
investigations
laboratories ary
with
data
since
of the
the goal
these
of obtaining
Fe-S proteins
the case of the -S. platensis the amino ently
acid
sequence
carried
out
the groups
arrived
difference
noted
acid
residue
analysis --et al. these
did
not
residues
assumption of the
In a previous automated
sequence
used in
the sequence
peptide
2
(1) and our
Our sequence
9 clearly
Asp.
showed
that
the sequence
constant
in
it
is Asn.
by our
for
and both there
showed
layer
of residues
the various
laboratory,
study
was one
that
amino
chromatographic In addition,
Wada
86 and 87 but
algal-plant complete
In
and independ-
However,
Our thin
relics.
approaches
simultaneously
the sequence.
was shown to be correct
S. platensis
was found
were
in several
and evolution-
to be ancient
same sequence.
determine were
thought
the
than
progress
in
two different
Wada --et al.
9 is Asn rather
of residue
are
determination
at nearly
are
structure-function
ferredoxin,
by both
in
ferredoxins
assumed
ferredoxins. sequence
This
investigations
ferredoxin2. report
from our
determination
(Z),
was described.
determination
to be a suitable
laboratory
of the
modification
for
This
a modification technique
S. platensis the sequence
of the again
ferredoxin
was
and
determination
T3.
The studies meeting in corrected is now in published experimental
reported here were completed in September 1975. At a Dec. 1975, Matsubara indicated that residue 9 has been to Asn. Thus, the sequence determination by the two groups complete agreement. However, it is important to have verification of the sequence, especially when different approaches have been used.
764
of
Vol. 69, No. 3, 1976
The amino siderable were
acid
homology
noted
sequence with
ferredoxins laboratory
AND BIOPHYSICAL
of the -S. platensis
the 2. maxima
in positions
and algal from our
BIOCHEMICAL
9, 52,
ferredoxin.
57, and 90.
have been compared (9)
and need not
RESEARCH COMMUNICATIONS
ferredoxin Only
four
The sequences
and discussed
be discussed
shows condifferences
of the other
in a previous
piant report
here.
ACKNOWLEDGEMENTS Research was supported in part by Grant GM 16784 and GM 16228 the National Institutes of Health, Grant GB 43448 from the National Science Foundation and the U.K. Science Research Council.
frvm
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 3. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Wada, K., Hase, T., Tokunaga, H., and Matsubara, H. (1975), .-FEBS Letters 55, 102. Tanaka, M., Haniu, M., Yasunobu, K.T., Rao, K.K., and Hall, D.O. (1975), Biochemistry 14, 5535. in Iron-Sulfur Proteins Yasunobu, K.T., and Tanaka, M., (1973), (Lovenberg, W., ed), Vol. II, pp. 27-130, Academic Press, New York. Matsubara, H., and Sasaki, R.M. (1968). J. --Biol. Chem. 243, 1732. Benson, A.M., and Yasunobu, K.T. (1969),-L. Chem. 244, 955. --Biol. Keresztes-Nagy, S., Perini, F., and Margoliash, E. (1969), J. Biol. Chem. 244, 981. Rao, K.K., and Matsubara, H. (197G), Biochem. --Biow. --Res. -Commun. 38, 500. Sugeno, K., and Matsubara, H. (1969), 2. -Biol. 2979. --Chem. s, Tanaka, M., Haniu, M., Zeitlin, S., Yasunobu, K.T., Evans, M.C.W., Rao, K.K., and Hall, D.O. (1975), Biochem. Biophys. 64 -Res. --Commun. --.I 399. Hall, D.O., Rao, K.K., and Cammack, R. (1972), Biochem. Biophys. --P.es. Commun. 5, 798. Wang, S.-S., and Carpenter, F.H. (1965), 2. --Biol. Chem. 3, 1619. Vesterberg, 0. (1971), Methods Enzymol. 2, 389. Edman, P., (1970), in Protein Sequence Determination (Needleman, S.B., ed), p. 211, Springer-Verlag Berlin, He-g, and New York. Edman, P., and Begg, G. (1967). Eur. J. Biochem. I, 80. --Braunitzer, G., Schrank, B., and Ruhfus, A. (lq70), Hoppe-Seyler's -Z. Physiol. Chem. 351, 1589. Pisano, J.J., and Bronzert, T.J. (1969), J. Biol. s. 235, 5597. Van Orden, H.O., and Carpenter, F.H. (1964), -Biochem. -Bio&. -Res. Commun. 14, 399. Ambler, R.B. (1967), Methods Enzymol. 11, 436. Bradbury, J.H. (1958)xem. J. 68, 475. Spackman, D.H., Moore, r and Stein, W.H. (1958), Anal. Chem. 30, 1190.
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