May 30, 1986 - 6 1987 by The American Society of Biological Chemists, Inc. Vol. 262, No. 3, , hue of January 25. pp. 1001-1009,1987. Printed in U.S.A..
Vol. 262, No. 3,, h u e of January 25. pp. 1001-1009,1987 Printed in U.S.A.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 6 1987 by The American Society of Biological Chemists, Inc.
The Primary Structure of Iron-Superoxide Dismutase from Photobacterium leiognathi* (Received for publication,May 30, 1986)
Donatella BarraSP, M. Eugenia SchininaQ, WilliamH. Bannisterll, Joe V. Bannisterv, and Francesco BossaS From the SDipartimento di Scienze Bimhimicheand Centro di Bwlogia Molecolare d e l Consiglw Nazwnale delle Ricerche, Universit(r La Sqpienza, 00185 Roma, Ztdy and the llBwtechnology Center, Cranfield Institute of Technology, Cranfield, Bedfordshire MK43 OAL, Great Britain
The complete amino acid sequence of iron-superox- as an enzyme containing copper and zinc (1, 2). Further Photobacterium leiognathi was de- investigations revealed the presence of manganese- and ironide dismutase from termined. The sequence was deduced following char- containing superoxide dismutases (3, 4). The superoxide disacterization of the peptides obtained from tryptic, chy- mutases containing either manganese or iron are considered motryptic,and Staphylococcus aureus V-8 protease to be a distinct class from the copper- and zinc-containing digests oftheapoprotein.Theaminoacidsequence isoenzymes. Structure-function relationships in the copper/ listed below is made up of 193 residues. It is the first zinc-superoxide dismutases have been the subject of various complete sequenceto be determined for an iron-super- reviews in recent years (5-9). However, more detailed invesoxide dismutase. The iron-superoxide dismutase shows tigations of the manganese- or iron-containing isoenzymes the same order of homology with the manganese-su- are still required before structure-function relationships can peroxidedismutases as theseenzymesshowamong be established for this class of superoxide dismutase. themselves. No homology was observed with the copWhereas copper/zinc-superoxide dismutase is, with three perlzinc-containingclass of superoxide dismutases.
exceptions (10-12), a eukaryotic enzyme, the manganese enAla-Phe-Glu-Leu-Pro-Ala-Leu-Pro-Phe-Ala-Met-Asn-Ala-zyme is found in mitochondria and in bacterial species. The iron enzyme, which was initially thought to be a bacterial Leu-Glu-Pro-His-Ile-Ser-Gln-Glu-Thr-Leu-Glu-Tyr-His- enzyme, has recently been purified from three plant species Tyr-Gly-Lys-His-His-Asn-Thr-Tyr-Val-Val-Lys-Leu-Asn- (13-15). The latter enzyme is the form of superoxide dismutase isolated from anaerobic bacteria such as Desulfovibrio Gly-Leu-Val-Glu-Gly-Thr-Glu-Leu-Ala-Glu-Lys-Ser-Leudesulfuricans (16), Propionibacterium shermanii (17), and Glu-Glu-Ile-Ile-Lys-Thr-Ser-Thr-Gly-Gly-Val-Phe-Asn- Bacteriodes fragilis (18).The enzyme from B. fragilis can have the iron substituted by manganese without loss of activity Asn-Ala-Ala-Gln-Val-Trp-Asn-His-Thr-Phe-Tyr-Trp-Asn(18), and P. shermanii was shown to synthesize the same Cys-Leu-Ala-Pro-Asn-Ala-Gly-Gly-Glu-Pro-Thr-Gly-Glu- enzyme with either iron or manganese depending on the metal supply (19); but more generally, the metal requirement for Val-Ala-Ala-Ala-Ile-Glu-Lys-Ala-Phe-Gly-Ser-Phe-Ala-Gluactivity is specific. Crystallographic data have indicated that Phe-Lys-Ala-Lys-Phe-Thr-Asp-Ser-Ala-Ile-Asn-Asn-Phe- the manganese- and iron-superoxide dismutases are structural Gly-Ser-Ser-Trp-Thr-Trp-Leu-Val-Lys-Asn-Ala-Asn-Glyhomologs (20). However, whereas the amino acid sequences and an x-ray structure at 2.4-A resolution are available for Ser-Leu-Ala-Ile-Val-Asn-Thr-Ser-Asn-Ala-Gly-Cys-Pro-Ilemanganese-superoxide dismutases (21-25), only the x-ray Thr-Glu-Glu-Gly-Val-Thr-Pro-Leu-Leu-Thr-Val-Asp-Leustructure has been reported for the iron-superoxide dismutases (26, 27). A primary structure has not been reported for Trp-Glu-His-Ala-Tyr-Tyr-Ile-Asp-Tyr-Arg-Asn-Leu-Argany iron-containing superoxide dismutase. The amino acid Pro-Ser-Tyr-Met-Asp-Gly-Phe-Trp-Ala-Leu-Val-Asn-Trpsequence reported in this investigation is the first sequence of the polypeptide chain of an iron-superoxide dismutase. The Asp-Phe-Val-Ser-Lys-Asn-Leu-Ala-Ala results reported suggest that theiron- and manganese-superoxide dismutases may have the same ligands to themetal and The reduction products of oxygen (the superoxide radical show that the extent of sequence homology between the ironO;, the hydroxyl radical OH‘, andhydrogen peroxide (H,O,)) superoxide dismutase and the known amino acid sequence of are associated with oxygen toxicity in living cells. Organisms Escherichia coli (21), Bacillus stearothermophilus (22), yeast have developed defense mechanisms against these products. (23), and humanliver (24) manganese-superoxide dismutases The enzyme superoxide dismutase (EC 1.15.1.1) scavenges is of the same order as between the manganese-superoxide superoxide radicals by the following reaction: 20; + 2H+ -+ dismutases. Hz02
+ 02.
EXPERIMENTAL PROCEDURES AND RESULTS’
Superoxide dismutase was discovered nearly 2 decades ago
* This work was supported in part by a grant from the Minister0 della Pubblica Istruzioneand sponsoredby Consiglio Nazionale delle Ricerche Strategic Project “Biotecnologie.”The costs of publication of this article were defrayed in part by the payment ofpage charges. This articlemustthereforebeherebymarked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. 8 TOwhom correspondence should be addressed: Dipartimento di Scienze Biochimiche, Universiti La Sapienza, PiazzaleAldo Mor0 5, 00185, Roma, Italy.
Portionsof this paper (including“ExperimentalProcedures,” “Results,”Tables 111-X, and Figs. 3-6) are presented in miniprintat the end of this paper.Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 RockvillePike, Bethesda, MD 20814. Request Document No. 86M-1819, cite the authors, and include a check or money order for$7.60 per set of photocopies. Full size photocopies are also includedin the microfilm edition of the Journal that is available from Waverly Press.
1001
Iron-Superoxide Dismutase
1002
25
Ala-ehe-Clu-Leu-Pro-Ala-Leu-Pro-Phe-Ala-r*L-~n-A1a-Lcu-Clu-Pro-Hia-Ile-Ser-Cln-Clu-lhr-Lcu-Clu-~r-Hlr-~r-Cly-~a-Hla-Hlr-A.n-lhrT l --14-s2-b
""0,
c2
c1-1
T2
3-1
s3
I
I
-
50
Thr-
Tyr-Val-Val-Lys-Leu-Asn-Cly-Leu-Val-Clu-Cly-Thr-----Clu-Leu-Ala-Clu-Lya-Ser-~u~lu~lu-Ile-Ile----Ly~-------------------T3
"""-"
s5
14
+-S6-1
S7-I
"-
#
" " " " " " "
75
Am-Ala-Cly-Cly-Clu-Pro"--
Ser-Thr-Gly-Cly-Val-Phe-Asn-Asn-Ala-Ala-Cln-Val-Trp-Asn-His-Thr-Phe-Tyr-Trp-Asn-Cys-Lcu-Ala-Pro---------
-*
C56C"
"""""""""""""""S8---""""""""""""""""""
c7-
100
125
Thr-Cly-Clu-Val-Ala-Ala-A1a-Ile-Clu-Lys-Ala-Phe-Cly-Ser-Phe-Ala-Clu-Re-Lys-Ala-Lys-Phe-Thr-Asp-Scr-Ala-Ile-Aan-~n-P~-Cly-~r-~r""""-"""""~b T6 >t-T7+--?e"-
"_
..
"
"_
_. 1 50
Trp-Thr-Trp-Leu-Val-Lys-Asn-Ala-----Asn-Cly-~r---Lou-Ala-lle-Val-Asn-Thr-Ser-Asn-Ala-Cly~s-Pr~lle-Thr~lu~lu---------------
""""_" 4
""_
+b
TO
-
""""""""""""ell"""""""""-" Cllb """""-Slz"""""""""""""~
1 b
175
--~ly-Val-Thr-Pro-Leu-Lcu-Thr-Val-Asp-Leu-Trp-Clu-His-Ala-Tyr-Tyr-Ile-Asp-Tyr-Arg-Asn-Lcu-Arg-Pro-Ser-Tyr-~t-A.p-Gly-Phe-Trp-Ala-
"""""_
"_
I
c1z
-4)
-4
513
-______-
- - -" - - -"" #
+,
-
T10-
g-Cl3 C14
""I
-SI4
"""""
zoo Leu-Val-Asn-Trp-Asp-Phe-Val-Ser----Lys-Asn-Leu-Ala-Ala l-Tll+
-C15" b
C17 C16-
--- - -
4
+ 4
FIG. 1. Complete amino acid sequence of iron-superoxide dismutase from P. leiognathi. -, extent of the various fragments used to construct the sequence; - - -, sequences inferred from amino acid compositions. T,tryptic peptides; C, chymotryptic peptides; S, S. aureus protease peptides. Gaps in the sequence have been introduced to obtain maximal homologies with manganese superoxide dismutases (see Fig. 2). DISCUSSION
The complete amino acid sequence of iron-superoxide dismutase from Photobacterium leiognathi is given in Fig. 1. The primary structure is made up of 193 amino acids, and the composition deduced from the sequence agrees with that obtained for the protein following acid hydrolysis (Table I). The sequence was deduced by isolation and characterization of the complete set of tryptic peptides, which were aligned by overlapping peptides obtained following chymotryptic digestion. Peptides isolated from a Staphylococcus aureus protease digest were useful in improving the structuralinformation for some regions and inproviding further evidence for the correct alignment of tryptic and chymotryptic peptides in two cases (T6 and T7, T7 and T8) where the overlap was of only 1 single amino acid residues. The extensive use of high-performance liquid chromatographic procedures on macroporous reverse-phase columns greatly facilitated the work necessary for peptide purification, as compared with our previous experience with the sequence determination of human liver manganese-superoxide dismutase (24). All the tryptic peptides terminated with either lysine or arginine with the exception of T11, thus implicating it as the COOH terminus of the protein, in accord with the results of carboxypeptidase digestion of the intactprotein. Further evi-
dence for the COOH-terminal sequence of the protein came from peptides c17 and S14, both terminatingwith a sequence not compatible with the specificity of the proteolytic enzyme used. A 39-residue NH2-terminal sequence of the protein was previously determined (31). The positioning of tryptic peptide T1 at theNH, terminus, followed bypeptide T2, was therefore obvious. In this NH2-terminalsequence, glycine wasreported to be presentin position 26; however, our results clearly indicate that this position is occupied by a histidine. This means that a histidine residue is present in this position for all the complete and partial amino acid sequences determined for manganese- and iron-superoxide dismutases (7, 31). The amino acid sequence of the iron-superoxide dismutase from P. leiognathi is compared with the published sequences of manganese-superoxide dismutases in Fig. 2. Gaps have been inserted to maximize the homologies whichare listed in Table 11. The iron-superoxide dismutase shows the same order of homology with the manganese-superoxide dismutases as these enzymes show among themselves. In contrast to the manganese-superoxide dismutases, where cysteine is confined to theeukaryotic forms, this residue is present inP. biognathi. Glycine residues, which often have a specific structural role in the folding of the polypeptide chain, are present at more
Iron-Superoxide Dismutase TABLE I Amino acid composition of P. leiognuthi iron-superoxide dismutase Carboxy- Recalculation Sequence Unmodified" methy,atedb from Ref. 28
Amino acid
Carboxymethylcysteine Aspartic acid Threonine11.2 Serine9.2 Glutamic acid Proline Glycine 20.9 Alanine 22.5 Half-cystine Valine Methionine 2.0 Isoleucine Leucine Tyrosine Phenylalanine Histidine 9.7 Lysine 9.3 2.8 Arginine 2.1 Tryptophan Total residues
2.0 21.8 12.9 9.8 19.1 8.0 13.7 23.1 2.2' 11.711.8 2.0 7.2 7.8 16.8 7.1 7.8 10.7 11.0 5.7 9.0 2.2 6.5 ND
19.822' 19.5 8.1 14.1
20.9 11.1 9.8 21.2 7.9 13.8 NW
9.9 0.9 6.8 16.2 7.5 10.3 5.4
15.9 6.1
6.28
13 11 lSd 8 14 23 2 12 2 8 17 8 11 6
2 7 193
Acid hydrolyses were performed on unmodified aposuperoxide dismutase for 24, 48, and 72 h. The values for threonine and serine were obtained by extrapolations to zero time of hydrolysis. Values of valine and isoleucine were from 72-h hydrolysates. Carboxymethylated aposuperoxide dismutase was hydrolyzed for only 24 h. Obtained as described under "Experimental Procedures." 5 aspartic acid and 17 asparagine residues. 16 glutamic acid and 2 glutamine residues. 'Determined as cysteic acid after hydrolysis in the presence of dimethyl sulfoxide (29). 'ND, not determined. Determined after hydrolysis with 4 N methanesulfonic acid (30). a
'
E.coli
1003
or less the same level and position in bothforms of superoxide dismutase, thus suggesting for this iron enzyme a threedimensional structure similar to thatdetermined for the manganese enzymes (25-27). The highest homologies between the five proteins listed in Fig. 2 occur in the 4-39 (near the NHz terminus)and in the 170-202 (near the COOH terminus) regions, where 14 out of 36 residues, i.e. 39%, and 13 out of 33 residues, i.e. 39%, respectively, are common in all the sequences. The recentdetermination of the x-ray structure of the manganese-superoxide dismutase from Thermw thermophilw at 2.4-A resolution has tentatively assigned His2', HisS3, Asp"j5, and Hid6' as ligands to the manganese (25). These align to Hisz6,Hiss1, Asp175, and Hid7'in the aligned sequences presented in Fig. 2. It is therefore clear that theligands to the manganese and iron are in identical positions in the primary structures of the two isozymes. The x-ray structure determined for two iron-superoxide dismutases has tentatively assigned residues 26, 69, 148, and 152 as ligands in E. coli iron-superoxide dismutase (26) and TABLE I1 Sequence homologies between iron- and manganese-superoxide dismutases Values are given as percentage of identical residues among the total residues aligned in Fig. 2. (Mn)
(Mn)
E. coli S. cereuisioe Human liver P. leiogmthi (Mn) (Fe)
B. stearothermoph49.8 47.8 39.3 59.9 ilus (Mn) 39.3 42.4 E. coli (Mn) S. cereuisiue (Mn)
38.8 42.4
Human liver (Mn)
34.1 35.0
/
60
"
:I
EL coli
----
Human l i v e r
120
B.8tearoth.
-
---
P.leiognathi
-
---
140
FIG. 2. Comparison of the amino acid sequences of B. stearotherrnophilus, E. coli, Saccharornycee cereuisiae, and human liver manganese-superoxide dismutases and P. leiognathi iron-superoxide dismutase. Boxes indicate positions a t which residues are identical.
K
1004
Iron-Superoxide Dismutase
residues 26,69,151, and 155as ligands inPseudomonas ovalis iron-superoxide dismutase (27). In the absence of a complete primary structure forthese two enzymes, it is not possible to determine the position of the ligands to the iron, with the exception of position 26. However, the residues liganding to the manganesealignwithironsequences.Whengapsare excluded from the P. lewgnathi sequence, the ligands to the ironare in positions 26,73,157,and161. These compare favorably with the positions so farinterpretablefrom the crystal structure in view of the fact that the iron-superoxide dismutases from E. coli and P. ovalis appear to be slightly smaller proteins (4, 32). REFERENCES 1. McCord, J. M., and Fridovich, I. (1969)J. Biol. Chem. 244,60496055 2. Carrico, R. J., and Deutsch, H. F. (1970)J. Biol. Chem. 245, 723-727 3. Keele, B.B., McCord, J. M.,and Fridovich, I. (1970)J. Biol. Chem. 245,6176-6181 4. Yost, F. J., and Fridovich, I. (1973)J. Biol. Chem. 248, 49054908 5. Steinman, H. M. (1983)in superoxide Dismutase (Oberley, L., ed) Vol. 1, pp. 11-68,CRC Press Inc., Boca Raton, FL 6. Bannister, J. V., and Rotilio, G. (1984)Deu. Biochem. 26, 146189 7. Parker, M. W., Schinina, M. E., Bossa, F., and Bannister, J. V. (1984)Inorg. Chim. Acta 91,307-317 8. Fielden, E. M., and Rotilio, G. (1984)in Copper Proteins and Copper Enzymes (Lontie, R., ed) Vol. 11, pp. 27-62,CRC Press Inc., Boca Raton, FL 9. Parker, M.W., Bossa, F., Barra, D., Bannister, W.H., and Bannister, J. V. (1986)in Superoxide and SuperoxideDismutase in Biology, Chemistry and Medicine (Rotilio, G., ed) pp. 237245,Elsevier/North-Holland Biomedical Press, Amsterdam 10. Puget, K., and Michelson, A.M. (1974)Biochem. Biophys. Res. Commun. 58,8304338 11. Steinman, H. M. (1982)J. BWL Chem. 257,10283-10293 12. Steinman, H. M. (1985)J. Bacterial. 162,1255-1258 13. Salin, M.L., and Bridges, S. M. (1982)Plant Physiol. 69, 161165 14. Kwiatowski, J. K., Safianowska, A., and Kaniuga, Z. (1985)Eur. J. Biochem. 146,459-466
OF IRON SUPEROXIDE DISMUTASE FROM THE PRIMARY STRUCTURE PHOTOBACTERIUM LElOSNATHl
BY
DONATELLA BARRA, M.EU6ENIA BANNISTER AN0 FRANCESCO BOSSA.
SCHININA'. WILLIAM
H . BANNISTER. JOE V .
EXPERINENTAL PROCEDURES
was ldognathi cultured Photobacterium Materials. a c c o r d i n g Pt ou g e t and M i c h e l r o n ( 3 3 1 and i r o n s u p e r o x i d e d1smuta.e was p u r i f i e d according t o Y a m a k w a ( 3 2 1 and B a n n i s t earnBd a n n i s t e r ( 3 4 1 . P r o c e d u r ef o sp rr e p a r a t i oot nhfaep o p r o t e i n and s u b s e q u e n t c a r b o x y m e t h y l a t i o n were I S d e s c r i b e d f otrh e human manganese enzyme CDIIC . arboxypeptidase (241. T r y p s i n( c o d e TRTPCKI, c h y m o t r y p s i n( c o d e A (COAPISI were f r o m N o r t h i n g t oBni o c h e m i c a l Co.; c a r b o x y p e p t i d a s e Y from Boehringer GmbH; S t a h l o c o c c ~ saureus V-8 p r o t e a s e nd p e p s i n from S i g mCah e m i c a l L a . ; p t k m o l y r i nf r o m Mcrck. l 0 d o l 2 - ~ ~ C l a c e t a t e w a s R at fhdr eioomc h e m C iecAnam tleer .r r h a m . Bucks. U.K.; g u a n i d i n e - H C 1 w a s r e c r y s t a l l i z e df r o mm e t h a n a l . ( 2 0 ngl E n z y m act il ce a v a g eSs- .e a r b o x y m e t h y l a taepdo p r o t e i n w a s suspended in 1.0 m1 O f 0 . 1 M ammonium b i c a r b o n a t e and i n c u b a t e d a t 37'C f o r 2 h w i t ht h ea d d i t i o n O f t w oa l i q u o t s O f t r y p s i na t 0 and 1 h. The enzyme t os u b s t r a t e ( E I S I r a t i o w a s 1 / 4 0 , A second 1 m p 1 e O f S - c a r b o x y n e t h y l s t e da p o p r o t e i n( - 1 0 0 ngl was suspended i n 1 0 n l O f 0.1 M m m o n i m b i c a r b o n a t e and i n c u b a t e da t 37' C f o r 3 h w i t ha d d i t i o n O f 0 and 2 h . The E I S r a t i o was 1140. A t w oa l i q u o t $o fc h y l o t r y p r i na t t h i rsda m p loeSf - c s r b a x y m e t h y l a t eadp o p r o t e i n ( 3 0 n g l was d i g e s t e d f o r 4 h w ti6thh1e" - s p e c i pf irco t e afsr eo m a u5 r. e u s under i d e n t i c a l c o n d i t i o n s . The E/S r a t i o w a s 1 / 5 0 .
15. Duke, M.V., and Salin, M.L. (1985)Arch. Biochem. Biophys. 243,305-314 16. Hatchiken, E.C., and Henry, Y.A. (1977)Biochemie (Paris) 59, 153-161 17. Meier, B., and Schwartz, A.C. (1980)Deu. Biochem. 11A, 160167
18. Gregory, E. M., and Dapper, C. H. (1983)Arch. Biochem. Biophys. 220,293-300 19. Meier, B., Barra, D., Bossa, F., Calabrese, L., and Rotilio, G . (1982)J. Bwl. Chem. 257, 13977-13980 20. Stallings, W. C., Pattridge, K. A., Strong, R. K., and Ludwig, M. L.(1984)J. BwL Chem. 259,10695-10699. 21. Steinman, H. M. (1978)J. Biol. Chem. 253,8708-8720 22. Brock, C. J., and Walker, J. E. (1980)Biochemistry 19, 28732882 23. Ditlow, C., Johansen, J. T., Martin, B.M., and Svendsen, I. B. (1982)Carlsberg Res. Commun. 47,81-91 24. Barra D., Schininl, M. E., Simmaco, M., Bannister, J. V., Bannister, W. H., Rotilio, G., and Bossa F. (1984)J. Biol. Chem. 259,12595-12601 25. Stallings, W. C.,Pattridge, K. A., Strong, R. K., and Ludwig, M. L.(1985)J. Biol. Chem. 260, 16424-16432 26. Stallings, W. C., Powers, T. B., Pattridge, K.A., Fee, J. A., and Ludwig, M. L. (1983)Proc. Natl. Acud. Sei. U.S. A. 80,38843888 27. Ringe, D., Petsko, G. A., Yamakura, F., Suzuki, K., and Ohmori, D. (1983)Proc. Natl. Acud. Sci. U.S. A. 80,3879-3883 28. Vanopdenbosch, B., Crichton, R.R., and Puget, K. (1977)in Superoxide and Superoxide Dismutases (Michelson, A.M., McCord, J. M., and Fridovich, I., eds) pp. 199-205,Academic Press Inc., Ltd., London 29. Spencer, R. L., and Wold, F. (1969)A d . Biochem. 32, 185-190 30. Simpons, R. J., Neuberger, M. R., and Liu, T. Y. (1976)J. Biol. Chem. 251,1936-1940 31. Harris, J. I., Auffret, A. D., Northrop, F. D., and Walker, J. E. (1980)Eur. J.Biochem. 106,297-303 32. Yamakura, F. (1976)Biochim. Biophys. Acta 422, 280-294 33. Puget, K.,and Michelson, A.M. (1974)Biochemie (Paris) 56, 1255-1267 34. Bannister, J. V., and Bannister, W. H. (1984)Methods Enzymol. 105,88-93 35. Hartley, B. S. (1970)Biochem. J. 119,805-822 36. Giglio, J. R. (1977)A d Biochem. 82,262-264 37. Simmaco, M., Barra, D., and Bossa, F. (1985)J. Chromutogr. 349,99-103 38. Hayashi, R., Moore, S., and Stein, W. H. (1973)J. Bwl. Chem. 248,2296-2302
P e p t i dp eu r i f i c a t i o n . The c h y m o t r y p t i c p e p t i d e s w e fr ier s t by g e l - f i l t r a t i o n an a S e p h a d e6x- 2s5u p e r f i nceo l u m n 5% a c e t i ca c i d .M o n i t o r i n go f column e f f l u e n t 1 2 . 5 ~ 1 1 0 c m l e l u t e dw i t h *IS p e r f o r m ebdy measuring t r a n S @ + i t t a n ca2et5 4 nn [ F i g . 3 1 and by t hliany ce hr m m a t o g v a p h y on c e l l u l e rpel a t e s I M e r c k Ab; s o l v e n t : n - b u t a n a l l a c eat icci d l w a t e r l p y r i d i n1e5./ 3 / 1 2 / 1 0 . v l v ) aol fi q u o t s spaced t u b e r ,t og e ti n f o r m a t i o n on t h ec o m p o s i t i o n f r o ma p p r o p r i a t e l y Of t h ed i f f e l e n tf r a c t i o n s i n t e r m s O f number and m o u n ot pf e p t i d e s . wdint h inhydrin and E h r l i cr eh a g efnotr The p l a t e s were s t a i n e was a c h i e v e d b yh i g hp e r f o r m a n c el i q u i d t r y p t o p h a nF. i n aplu r i f i c a t i o n Chromatography (HPLCl u s i n g a Beckman n o d e l 332 i n s t r u m e n t . on macroporous r e v e r s e - p h a s e co1umn1 ( ~ r e w n l eLea b s , quap pore R P - 3 0 0 ; 0 . 7 ~ 2 5 cm, 1 0 pml e l u t e dw i t hg r a d i e n t so f 0 t o 701 a c e t o n i t r i l e i n 0 . 2 X t r i f l u o r o a c e t i c a c i d . a t a f l o wr a t eo f 3.0 m l l m i n E . l u t i o no f was m o n i t o r e d on d B e c k m a n 1 6 s5 p e c t r o p h o t o m e t ear t 220 t h ep e p t i d e s and 280 nm. Monitoring O f p u r i f i c a t i o n O f S-carboxy~cthylcyrtLlnc C o n t a i n i npge p t i d e s was p e l f o r m ebdcyo u n t i ntghTea d i o a c t i r i t y O f s u i t a b l ea l i q u o t s O f t h ef r a c t i o n sf r o mt h ec o l u m np u r i f i c a t i o ns t e p s a n d b ya u t o r a d i o g r a p h y Of t h et h i nl a y e rp l a t e r . The t r y p t i cd i g e s t O f S - c a r b o x y m e t h y l a t e da p o p r o t e i n was d i l e c t l y was p u r i f i e d by HPLC. F i r s t . a p i l o ta n a l y s i s O f 40 m o l O f t h ed i g e s t p e r f o r m e(dF i q . 41. i n o r d e r t o c h o o steheel u t i oCn o n d i t i o nbse l t s u i t e d f o r p e p t i d ep u r i f i c a t i o n . Then. t h e renaioing O f t h ed i g e s t was i n j e c t e d i n s e v e r aal l i q u o t sa. b o u2t 0 0m oel a c h . on the preparative
fractionated
COI".".
S i m i l aptrehl ype .t i od be tsa i an dfetideg re s t i o n O f the S - c a r b o x y m e t h y l a t epdr o t e iwni t h 5 . auleus P I O t e a saenwdh i c h were soluble i n 0.21 trifluoroacetic a c i d "ere d i r e c t l iyn j e c t e d on t h e HPLC c o l u m n( F i g 5. 1 t; h ei n s o l u b l ef r a c t i o n vas s o l u b i l i z e dw i t ht h e a d d i t i o n O f g u a n i d i n e - H C 1 I 6 M f i n a l 1 and t h e np u r i f i e db y HPLC ( F i g . 61. A n a l y t i c tael c h n i q u e Q s .u a n t i t a t i v e a m i n o a c ai dn a l y s e s were c a r r i e d Out on 0 . 5 - 2m o l O f peptidh eydrolyzed i n 200 p l Of 6 M H C l . c o n t a i n i n g0 . 1 sp h e n o l a . t 11O'C f o r 24 h. The amino acid composition ohf y d r o l y s e d peptides and o f t h e p r o t e i n w a s d e t e r m i n e d
-
Iron-Superoxide Dismutase u s i n g an L I B 4 4 0 0i n s t r u m e n et q u i p p e dU I t h 1 Spectra P h y s i c sS y s t e m I computin I ngt e g r a t o r . The C - t e r m i nsael q u e nt ocp h fer o t e i n was a n a l y s e db y~ a ~ b o ~ y p e p t i d a s e A d i g e s t i o ni n 0.1 M ammonium b i c a r b o n a t e at 3 7 ° C f o r 3 h.The E/S ratlo c o n t a i n l n g0 . 1 %1 0 d i md o d e c y sl u l f a t e *as 1/20, The amino a c i ds e q u e n c e s O f p e p t i d e s was m a i n l yd e t e r m i n e db yt h e d a n r y l - ~ d m taenc h n i q d u e s c r i bH b e yda r t l e y (351. D a n r y l - T r p war i d e n t i f i e da f t e r 5 h - h y d r o l y r i ~ a t 11O'C w i t h 4 N n e t h a n e r u l f o n i ca c i d containlng 0.2% 3 - ( 2 - a ~ i n o e t h y l l i n dO o lct eho,enrd i t iboeni sn g i d e n t i c a lt ot h o r ed e s c r 7 b e di n 1 3 6 1 . The a m i d a t i o ns t a t e or 6f 1 " and Asp were a s s i gdnbi reyiedcet n t i f i c a t bi oy n H P L C Of t h e phcnylthiahydantoin d e r i v d t i v ~ vs e l e a s e d u ~ i n g dansyl-Ednan sequence a n a l y s i st;h e same procedure M I S used f o tr h ei d e n t i f i c a t i o n Of t h e p h e n y l t h 7 a h y d a n t o idne r i v a t i v e O f c l r b o x y ~ e t h y l c y l t e i n e0 7 1 . I n S o m e d i g e s t i o nw i t h c a r e r . f u r t h esr e q u e n c ien f o r m a t i o n w a s O b t a i n e da f t e r ~ a r b o x y p c p t i d a l e Y I 3 8 1 and i d e n t i f i c a t i o no ft h ea m i n oa c i d sr e l e a s e d baym i naoc ai dn a l y s i Y s .h e rnee c e s s a r ay l. i q u o pot $ef p t i d e were furthe f rra g m e n t ewdi at hp p r o p l i a t e plocedules. Subdigertiow n ri t h t h e r n o l y r i n or w i t h 5. luleus p r o t e a s e were p e r f o r m ei nd 0.1 H an E I Sr a t i o = 1 / 3 0f o r 1 h a t 37'C. P c p t ( c m m o n i mb i c a r b o n a t e* ? t h d i g e s t i o n * a s p e l f o r m e di n 51 f o r m i ca c i dw i t h an E I S r a t i o = 1 1 5 0f o r 1 h a t 31°C a n d f r a g m e n t a t l e n sw i t h CNBr were p e r f o r m e d i n IOX f o r m i c 24 h ti h ep e p t i d e 10 CNBr r a t i o w a s 1 : l a c i d a t r o o m t e m p e r a t u r ef o r 1w:Wl. Products O f t h eSscec o n d a l y fragmentation p r o c e d u r e s Were pulified by HPLC O n a n a l y t i c arle v e r r e - p h a r e COIU~III (BloWnlce Labs. AquapQre RP-300; 0 . 4 6 ~ 2 5 cm. 1 0 11.1 e l u t e dw i t ha c e t o n i t r i l eg r a d i e n t s i n 0 . Z X t v i f l u o r o a c e t i ca t i da t a f l o wr a t eo f1 . 2m l l m i n .
1005
ClUIllllllrnlrnlN
Flg. 4. R e v e r s e p h a shei gphe r f o r m a n cl ei q u icdh r O n s t O 9 T a P h Y Of t h e t r y p t i cd i g e s o t a f pe-proteinC . o n d i t i o n so a f nalysis a r e r e p o r t e di n t h tee x t . The numbers above t h pe e a k rse f etrtoh ter y p t i pc e p t i d e s u b s e q u e n t l yf o u n di nt h e r e peaks.
were numbered Pneopm t i edpne cpT l tah id teuerse . r e t r o s p e c t i v e l ya c c o r d i n g t o t h e i rl o c a t i o ni nt h e sequence. s t a r t i n g f r ot N hme- t e r m i n uTsr.y p p t i ec p t i d e s a r e d e r ~ g n a t ewdi t h a T. C h y m o t r y p t ipce p t i d ews i t h a C, 5 . a u ~ e u s p ~ o t e a s e p e p t i d ews i t h a 5. fAr lal g m e no tbst a i nfer o sd umb d i g e s t i o n s a r e d e s i g n a bt eyd I second l a t t e r i n d i c a t i n g firit t hpea r e pn et p t i df oe l l e x ebdy i n d i c a t i n gt h ep r o t e o l y t i cm e t h o d ( 8 = CN8r; L = t h e r n o l y r i n ; 5 = 5 . aureus protease; P = p e p s i n 1 and numbered a c c o r d ti tn hog eG p e s i t l o ni nt h e sequence. s t a r t i n fgr o m thN e - t e r m i n u sF. oer x a m p l e . a f t~eur b d i g e r t i o n Of tthl ye p pt iecp t i d e T9 w i t h 5. aureur p r o t e a steh f .rea g m e nTt sO - S1l9. - 5 a2 n1 d 3-53 were p u r i f i e d and a n a l y s e d Iree T a b l e V and I V I .
f ,.
RESULTS
O f ptrhoet C e ianl.b o x y p e p t i d a s e A C - t e r msi n ea q lu e n c e t h~ea l b o x y m e t h y l a t cpdr o t e i n r e l e a r e d t hf oe l l O W i n g d i g e s t i o n Of m l n o acids 1.01 p e r mol of s u b ~ n i t l : Arn 10.61. A 1 l a1 . 5 1 and Leu 10.7).
no
P
acid Camposition O f t h et r y p t i c The amino Trypp t iec p t i d e s . T a b l e I l l . Amino a c i d s e q u e n c e s d e t e r m i n e d are p e p t i d e si sr e p o r t e di n rvnmsr1red Ti na b l e IV. I n some c a s e s . s u b d i g e s t i foonl l o w be yd and a n a l y s iotsh f gee n e r a t efdr a g m e n t s 111 n e C e s I I r y ; puliflcation a n a l y t i c a ld a t a O f t h e s ef r a g m e n t s a r c r e p o r t e di nT a b l e s I V and V . Chymotrypp t i cc p t i d eArn. a l y t i cdaal t a on t C h eh y m o t r y p t i c p e p t i d e sw h i c hg a v ei n f o r m a t i o nu s e f uflo C r o m p l e t i o notfh es e q u e n c e a r c r e p o r t e di nT a b l e s V I and V I I . A n a l y t i c adl a t a on sone p e p t i d e s are i n c l u d e do n l y i n T a b l e V I f o rt h es a k e of C l a r i t y .s i n c et h e y were n o t u t i l l s efdoc ro n s t r u c t i ntgh e sequence. The sequence of sone c h y m o t l y p t i pc e p t i d e s w a s e l u c i d a t e da f t erru b d i g e s t i o nw i t h VlrlouI p r o t e o l y t i c e n z y m e s( T a b l e s V I 1 and V I I I I . protease peptides. A n a l y t i c a l d a t a on t h e p e p t i d e s o b t a i n e d a f t e r d i g e s t i o n w i tt hh e 5 . lureus p r o t e a s e 61"-specific &(.e r e p o r t e d i n T h b l e r I X and X . T h e r pe e p t i d e cs o v e r e tdh e n t i r e sequeno c ef t h e p r o t e i tnh. vs su p p o r t i nt hgc eo r r e c t n e tsahsf e on t hbea s oitshfree s u l tosb t a i n ewdi tt hh e s t r u c t u rper o p o s e d t r y p t i c a nC d h y m o t r y p t i pc e p t i d e sw. i t thh ee x c e p t i o ont fh ter a c t f r o mT h r 2 2 t o 61"24.
5.
auvcus
mtmn ~ ~ u r n t m ~ ~ Fig. 3. E l u t i o np r o f r l eo tf h eC h y m o t r y p t i cd i g e s t F l or w ate was 19.5 m l l Fh r. a c t i o n s Were verticaliner.
pooled
an Sephadex 6 - 2 5 . IS indicatb e yd
Elutmn nmmcrnln, Fig. 5 . Reverse p h ahsipegehl f o r m a nl ci qeuc ihdr o m a t o g r a p h y O f soluble p r o t e adsi eg e s t Of a p o - p r o t eCi no.n d i t i o n s Of a n a l y s i s arc r e p o r t e di nt h e t e x t . The numbers a b o v e t h e p e a k sr e f e r t ot h ep e p t i d eI u b s e q u L n t l yf o u n d i n t h e r ep e a k s .
-
Iron-SuperoxideDismutase
1006
TlgLE I I I I l i n o Acid CDlpoIitiD"l The c n p a r i t i o n f r a sequence analysis 1-1 1-29
T-2 30-37
T-3 38-51
1.011) 0.911) 0.9(11 4.8151 2.7131 1.1111 4.0141
1.011) 1.0111
0.911) 0.911)
7-4 52-59
0.711) 2.0121
2.9131
1.512)
1.8121 1.011 I 0.9(11
0.611) 0.8Ill 1.612) 1.612) 1.712) 0.811)
1.612) 1.111)
2.7(3)
3.614)
0.9111 1.9121 1.0111
0.9(11
0.911)
Of
7-5 66-109
Presence
Of
27.3
44.9
3.8141
1.011 I
2.012) 5.0151 6.8(71 2.6131
1.0111 1.912)
1-7 119-120
1-8 121-138
2.8131 1.5121 2.5131
1.011 I
0.9111 5.716) 3.5141 1.8121 3.013) 1.6121 3.00) 4.0141 2.4131 2.5131 3.6(41 2.5131
3.714)
1.0(11
2.1121 0.911) 1.011) 1.1(1) 1.5121 0.7(11 1.8121 0.911 I 1.5121
2.1121
1.0111
1.111) 1.011 I
1.011 I
121
0.9111 +
121
13.6
58.2
72.7
17.1
Thr
Ala
Ala
Phe
T1
1 2
1 3 7 4
T 5
- 15-L4 F K _ - F_ _ ___-.
T 6
1110-1181
A FG
T I
(119-120)
A K
T B
(121-138)
FTBSAINNFGSISYTWLVK)
A E
".__.__"_
T9
-
VOLWEHAYYlOYR
"""..""-
" 19-53
7-9 1-10 1-11 139-186 187-208 209-212
1.5(21
2.5131
tryptophan was indicated by absorbance at 280 nn.
S
1.011 I 0.8(11 0.711) 0.7111 0.911)
1.0(11 1.1111 0.8(11 1.612) 1.0111 1.0111
Ser
Leu
1-6 110-118
0.8111
57.138.2
His
Tryptic Peptides
1.011 1 4.715) 3.8141 1.111)
+
yield I N-terminal Alaresidue
Of
each peptide i s indicated by the nlnberr i n parentheses
0.811) + 11)
0.911) 0.711) + 12)
14.5
28.0
62.1
AXI
Arx
Asx
Iron-Superoxide Dismutase
1007
TABLE V h i n o k i d C o q o r i t i o nO f F r l p T r n t l Obtained a f t e r S u M i g e r t i O n The c - s i t i m
from sequence a n a l y s i s O f each p e p t i d e i s i n d i c a t e d
12-82 12-29
T5-LI 66-77
15-L2 78-82
1.0(1 I o m 1I 1.0(1) 4.0(41 1.011l
1.8121 1.5121 0.7(11 1.011)
1.011 I 0.9111
l.l(Il
2.0(21 2.1(2) 0.911 I
T5-L3
83-87
15-14 88-109
19-51 139-161
1.0(1), 0.9(1)
0.9111 3.7(4) 1.7(21 1.7121
0.7(11
1.0(11
2.7131 1.6121 2.8131
TO-SZ
79-53
167-178
179-186
1.0(11 1.4(21
1 .011 I
2.0121
1.0(11
3.0(31
0.9111
0.8(1 I
1.0(1 I 1.0111 0.81 1 I 1.6(21
1.5121 1.011 I 2.6(3) 2.9131
0.7(11 0.8(11
0.8111
110-02 194-208
1.0(1)
2.9131 0.8111 1.612) 0.9111
0.8(11 0.8(11 0.8(1)
710-81 187-193
0.9(11 1.011 I 1.111l
1.8121 0.911 I
4.0(51 0.9(11
0.6111 1.8(21 1.5121 1.8(2) 1.0(11
I.O(l1
Tryptic Peptides
Of
by t h e nunbers i n parentheses
0.9111 0.9111
1.6121 1.0111
1.0111 0.811 I
0.811
0.9(1)
+ (11
O.B(ll
+
(11
I
0.9(11 + I21
+ (1)
+ (11
mr
AIX
VI1
Phe
Le"
G~Y
ASX
His
AIX
AS"
TULE V I 1111110
k i d CO.POsitiOnl
Of
Chynotryptic Peptides
The c w q o s i t i o n f m sequence a n a l y s i s o f each p e p t i d e i s i n d i c a t e d b y t h e Peptide residue
C-1
C-2
1-9
10-25
C-3 26-34
C-3d 28-34
c-4 35-72
I.O(l1
0.9111
0.7(11
1.0111
1.011l 1.8(21 2.513) 1.5(21 4.6(51 1.0(11
C-3b 31-34
C-3a 26-30
C-3E 26-27
numbers i n Darentheser
c-5 73-79
C-6 80-84
C-7 85-111
1.0(1)
1.0111 1.9(21
0.9(11
1.0(1)
C-8 112-117
nor.
CnCyS
1.0111 2.0121
1 .011 I 0.9111 0.9111 4.1141 0.8lll
1.8121
1.7(21
ASP
Thr Ser
Glu Pro
G~Y Ala Val
0.8111 0.8111
1.8(21
T v
Phe Hi5 LYS
I .011 I
1.0111 1.0111
W e t
Ile Le"
0.9111 0.9111
3.8141 0.9111 2.8141
0.9(11
1.8(2) 0.6(11
0.8(1l
2.8(3)
1.5(2l
1.0(1)
1.0(1)
1.011)
1.0(11
0.0111
1.0111
1.0111
2.0121 1.011 I
Peptide lesidue
1.8121 0.9111
2.4131 +
37.2
Ala C-8a 112-114
C-8b 115-117
0.9111
Ill
3.0
5.9
1.2
5.0
8.3
33.1
35.9
10.2
His
His
His
His
GlY
Val
A%
A%
Trp
C-lob
C-11
CC- l-llbl a 134-135
C-10 C-101 122-133 122-129 130-133 134-171
0.9111 0.8111
136-171
2.112)
1.0111 + I11
22.8
C-9 118-121
0.8(1l 1.0(1)
0.9111 1.011 I 1.0111 1.0111 1.0111
0.7(11
1.7(2)
26.9
1.9121 2.8131 5.816) 0.9111
1.2121 3.5(41
2.0(21
f v
Total yield I N-terminal residueAla
3.0(31
(-12 C-12a 172-182 172-177 178-182
34.8 Gly C-12b
nor.
2.8131 0.8111 2.4131
1.011l
0.8(1) 4.014)
3.0131 0.9111
0.9111
1.7(21
1.011l 1.0111 0.9111
0.8111
1.0111
1.1111 0.8111
1.011 I
0.8111
0.9111
0.7111 0.9111 1.011)
1.0111
1.3121 1.6121 2.701 3.0131 2.4131 1.7121 2.7131
1.011)
I.O(ll
1.0(1)
0.811)
1.011 I
1.011l 1.0(1) 1.6(21 1.912) 1.6(2)
1.8(21
0.9(11
1.0111
1.0111
2.0121 + Ill
4111
1.0111 +I11
0.5111 +
Ill
+ +I Il l l
2.1
1.8
23.7
3.4
10.8
34.0
2.0
66.4
16.0 6.1
4.1
Gly
Ala
Lyr
Thr
Thr
Gly
Thr
Thr
Leu
Leu
Leu
C-13a 183-185
C-14 186-197
C-14a
C-l4b 194-197
C-15
(-16
residue
E-13 183-188
C-16a 198-202 200-210 200-202
C-17 203-212
C-17a 203-210
C-l7b 211-212
Asp
2.0121
1.0111
1.9121 0.7lll 1.0111 1.0111
0.7111 1.0111
1.5121 0.9(11
1.6(21 0.8111
1.7121 0.7111
0.6111
0.8111
0.7111
Peptide
SW PTO
G ~ Y A1 a Val net Ile Leu Tyr Phe LYS Arg
0.7111 0.8111 0.8111
Total yield% N-terainal I l ree s i d u e
189-193
1.0(1)
3.0131
1.0111
0.7(11 1.0(1)
0.9(11 1.0111 0.6111
2.012)
1.0(1) 1.0(1)
1.6:2)
l.O(Il
0.8111
0.8(1l
0.8111
1.011 I 1.011 I
1.0(1) 0.8(1)
0.9111
23.1
14.0
I.O(ll 0.9111
0.8111
1.6121 +
1.2
0.9(11
22.7
1.0(11
1.0(11
Trp
lle
1.0111 0.9(11 1.0111
3.8(4) 2.0121
20.7 Arg
Ill 3.0 Arg
0.9(11
0.7111 + I l l 1.0
+ ( l l 46.0
Val
Asx Val
13.0
Ala
+Ill
+Ill
1.2
2.8
XIA
1.0111
ASX
30.1 Ala
Glx
Iron-Superoxide Dismutase
1008
TABLE VI1 suI.llry o f Sequence Studies en Chynotryptic Peptides
.*"...._ .".. "._._...."....
A F E L P A L P F
c 1
11-91
c 2
110-251
A M N A L L P H ~ S Z E T L E Y
c 3
126-34)
HYGKHHNTV
C4
(35-721
"."""
""...." -- -..".-
"._".... -- . . - *-. -*
Y Y K L N G L Y E G T E L A L K S L E E I l K T S T G G V F
C4-S1
CI-SP
c4-54
c4-53
c 5
(73-791
NNAAQYW
C6
(80-841
NHTFY
c 7
185-111)
""*..
"." . . " . _ _.... ._ -
- -- * . _ _-_ _ _ " U N C L A P N A G G E P T ~ E V A A A I E K A F
C7-S1
*C7-L14-
C7-SZ
"".".""".*"_" C7-LZ
C7-L3
4
"".*
C 8
(112-1171
GSFAEF
c
9
(118-1211
KAKF
C 10
(122-1331
TOSAINNFGSSW
C 11
(134-171)
T W L Y K 0 A 8 l G S L A 1 Y 0 T S 0 A G C P I T Z Z G V T P L )
C l l b (136-1711
C 12
1172-1821
_.*,
"..""....-..-.."."_.-* . """.""...""_..__l___ "."""-.
-
L Y K N A H G S L A I Y N T S N A G C P I T Z E G V T P L
Cllb-P1
Cllb-PZ
LTVOLVEHAYY
C 13 1183-188)
__""
C 14
RNLRPSYMOGFU
1186-1971
L O Y R N L
"""".". ."._
C 15 (198-2021
ALVNY
C 16 (ZW-2101
v n u o ~ v s ( K n ~ ~
C 17
OFVSKNLAA
(203-2121
-*-_... -."-.."
The residues above t h e arrw 1-1 Were i d e n t i f i e d bydanryl-Edaandegradation. The sequence d c t e r n i n e d by carboxypeptidase I d i g e s t i o n i s i n d i c a t e db y arrws 1-1 above thecorrespondingTellduel. Subfra-nts o b t a i n e da f t e rd i g e s t i o nw i t h S. ~ U I ~ U I protease IS), pepsin IPI 01 t h e m l y r i n ( L l arc i n d i c a t e d by s o l i d l i n e s .
TllBLE VI11 h i n o Acid CMlpositions of F l a g r n t s Obtained a f t e l S u t d i g e l t i o n o f C h m t r y p t i c P e p t i d e s
The c-sition c4-54 c4-53 c4-s2 peptide C4-Sl residues
35-43
frm sequence a n a l y s i s o f each p e p t i d e i r i n d i c a t e d by t h e number I n parentheses
44-50
48-55
C7-S2 C7-S1 85-102 56-72 103-111 88-106 85-87 107-111 136-146 147-171
C7-Ll
C7-LZ
1.0(11
1.0111 0.7111
C7-L3
Cllb-PI Cllb-PZ
"OS.
1.0(11 CnCYS ASP Thr SW Glu
0.711l
1.0(1) 1.9121 1.8121
0.7(11
2.0121
3.0(3)
1.011l
1.0111 0.8111
61y
N-terminal Val vtsiduc
2.0(21
1.5121 0.8111
0.1111
0.911l 1.8121 0.911)
Leu
2.0121 1.8121 2.813) 4.1151 0.8111
1.011)
1.7121 1.1131
2.0121
1.0111
1.6131
Ile Le" Phe LYS TVP
1.9121
0.9(1) 0.9(1) 1.1(11
PVO
61; A1 a VI1
1.0(11 1.9121 1.0111
3.8141 0.911) 0.9(1)
1.011) 1.212)
l lVal e
1.0111
0.8111
0.911) 0.911) 0.9111
0.811) 0.9(1)
1.1111
+ I11
+ I11
Trp
Trp
Leu
1.0111 1.0(11 1.0111 2.1121
0.011 I 0.9111
1.0(11
I lA11 e
Leu
1 .011 I 1.7121 2.5131 0.9111 1.7121 1.6121
2.0121 1.8121 1.4121 1.512) 0.9111
Iron-Superoxide Dismutase
1009
TbBLE IK
of 5 . aweus Peptides
hino kid C-mitiom
from sequence analysis O f each p p t i d e i s indicate4 by the nwberr i n palentheses
The c-sition Peptide 5-1 residue 44-47 25-43 11-21 4-10 1-3
5-25-5
5-3
5-7 48-55
5-6
5-8
56-10?
5-9 103-100
5-10
5-11
109-116 117-130
5-12 131-161
5-13 167-178
5-14 179-212
1.0111 1.4(21
6.0(6)
"OS.
0.81 11 4.8(5) 3.5(41 0.9111 3.0131 2.1121 4.7(51 4.0(41 1.712)
1.0(1) 2.0(21 0.9(1) 1.9121
0.7(1) 2.7(31 0.9111
2.0121
1.0111
1.0111
1.0(1) 1.8121
1.0(11
0.0111 1.0(11
0.9111 2.0131
0.9(1)
1.011)
2.3131 0.8(11 0.8(11
1.9121
1.3(2) 1.111)
1.7121
0.8(11 1.8(21
Presence
Of
1.0111
1.0(1)
1.0111
1.1(11
2.7(31 1.7121 2.0(21 0.9(11
4.0(41 2.5131 3.2141 1.0121 0.6111 2.1121
1.612) 1.1(11 0.6111
1.0111
3.0131
1.011 I
0.8(1)
1.7(21
1.5121
1.4121 1.7121
2.3131
1.7121
1.0111
0.8111
Total yield% 16.7 N-terminal residue 1\11
0.9(11
0.8111
2.5131 0.9111
0.9(11
3.0131 1.011 1 0.9111
50.7
21.7 Leu
k Gly t
0.8111
2.5131 1.9121
26.1
35.0 12.2 31.3
Tyr
Leu
tryptophan was indicated byabsorbance
0.811 I
0.9111
0.9111
Ile
29.8
5.2
Val
Lyr
18.3 Phe
4.7
16.6
13.2
Scr
Gly
His
a t 200 nm.
TIBLE
X
S u m m y of Sequence Studies on 5 . auIeus ProteasePeptides
5 1
(1-31
AF E
*..
.._.-__
5 14-101 2
L PA L PF A
...""".-
53
111-211 R N A L E P H I S Q E
5 5
125-43)
5 6
144-471 G T E
S 7
(48-551
5 8
(56-1021
_"......"""
Y H Y G K H H N T Y V V K L B I G L Y E I
..""..
L A E X 5 L E E
..*
I I K ~ T S T G G V F 0 0 A A l V Y 0 H T F ~ U 0 C L A P 0 A G G I PTGEl
I "."_
5 9
1103-1081 VAAA
5 10
1109-1161 K A F G 5 F A E
5 11
(117-1301
F K A X F T 0 5 A I N N F
512
1131-1611
S S Y T W L V K N A 0 ~ G S L A I V 0 T 5 0 A G C P l T l E 1
5 13
1167-1781 G V T P L L T V ( 0 L Y E)
514
(179-2121
E
"".... "."."."._.
G
.-....-__._ _.".." .""
0.6111 1.1111 4.2(41 1.9121 1.0111 1.011 I 3.0131 3.5141 1.8121 1.0111 1.0111 1.8121
H A Y Y I l 8 Y R 0 L R P S Y W B G F Y A L V 0 U 0 F ~ 5 K 0 L A A l
