King, T. E. (1982) in Function of Quinones in Energy Conseruing. Systems ... 192,1121-. 20. Wakabayashi, S., Matsubara, H., Kim, C. H., Kawai, K. & King,. 21.
Vol. 260, No. 1. Issue of January 10,pp. 337-343,1985 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.
Complete Amino Acid Sequenceof the Ubiquinone BindingProtein (QP-C), a Protein Similar to the 14,000-Dalton Subunit of the Yeast Ubiquinol-Cytochrome c Reductase Complex* (Received for publication, May 2, 1984)
Sadao WakabayashiS, Toshifumi TakaoQ, Yasutsugu ShimonishiQ, Seiki KuramitsulI, Hiroshi MatsubaraS,Tsing-ying Wangll ,Zhen-ping Zhangll, and TsooE.King11 From the +Department ofBiology, Faculty of Science, Osaka University, Toyomka, Osaka 560, Japan, the §Peptide Center, Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan, the llDepartmentof Medical Chemistry, Osaka Medical College, Takatsuki, Osaka 569, Japan, and the (1 Department of Chemistry and Laboratory of Bioenergetics, State University of New York at Albany, Albany, New York 12222
The amino acid sequence of the ubiquinone binding of the polypeptides was performed by photoaffinity labeling protein(QP-C)inthecytochrome b c l region of the of the cytochrome bcl complex with arylazido ubiquinone mitochondrial electron transfer chain was determined derivatives (8).Isolation of QP-C and reconstitution of the by analysis of peptides obtained by cyanogen bromide ubiquinone-cytochrome c reductase have also been accomcleavage and staphylococcal protease digestion of suc-plished (9). Chemical characterization of the ubiquinone bindcinylated derivatives. It was found to consist of 110 ing protein is thus vital for understanding electron transfer aminoacidresiduesand its aminoterminustobe in the ubiquinone-cytochrome c reductase complex. blocked byan acetyl group, as determined bymass This paper describes the first complete amino acid sequence spectrometry of the amino-terminal peptide and a comof one of the ubiquinone binding proteins, QP-C, presents the parison with peptides chemically synthesized on high- remarkable features of the primary structure, andpredicts the performanceliquidchromatography.Themolecular secondary structure. We compare this sequence with that of weight of this ubiquinone binding protein including the 14-kDa subunit of the yeast ubiquinol-cytochrome c rethe acetyl group was calculatedto be 13,389. The ductase complex deduced from the nucleotide sequence and predicted secondary structure of QP-C has a-helical content of about 50% and QP-C was classified as an speculate on the functions of these proteins. + 8“ protein.This is the first reportde“all-a” or MATERIALS AND METHODS AND RESULTS’ scribing the amino acid sequence of the ubiquinone binding protein. A comparison of this sequence with Three cyanogen bromide peptides were aligned by overlapthat of the 14-kDa subunitoftheyeastubiquinolping staphylococcal protease peptides so that the complete cytochrome c reductase complex from the nucleotide amino acid sequence of QP-C could be determined as shown sequence showed thesetwo sequences to be quite simin Fig. 1. The detailed results appear in the Miniprint Supilar. plement. The sequence was found to consist of 110 amino acid residues with no cysteine. The amino terminus is acetylated. The molecular weight of the sequence including the Nterminal acetyl group was calculated to be 13,389. Ubiquinone is an essential component of the mitochondrial electron transfer system (1, 2). It exists as a hypothetical DISCUSSION mobile component is molar excess compared to other electron Until QP-C was isolated recently, an understanding of its transfer components between electron transfer complexes (1). Recent studies at our laboratory have shown some of the molecular properties was made possible primarily through a ubiquinone to be bound to specific proteins which stabilize study of the cytochrome bcl complex. The molecular weight ubisemiquinone radicals (3). At least three different ubiqui- of the ubiquinone binding proteins was estimated to be about none binding proteins, QP-S,’ QP-C, and QP-N, have been 37,000 and 17,000 by photoaffinity labeling, suggesting the found in the mitochondrial respiratory chain (4, 5). Isolated ubiquinone binding sites to be cytochromes b (8). However, QP-S is capable of reconstituting succinate ubiquinone reduc- isolated QP-C, which restored the ubiquinone-cytochrome c tase with soluble succinate dehydrogenase (6). In the cyto- reductase activity and recovered the EPR signal of QP-Cchrome bcl region, the existence of another ubiquinone bind- deficient cytochrome bcl complex, showeda molecular weight ing protein, QP-C, was demonstrated (3,7), andidentification of 15,000 and was without any heme prosthetic group (9). Whether this QP-C and the smaller cytochrome b are the * This work was supported in part by Grant-in-Aid 58470131 for same polypeptide is not clear since no information on the Scientific Research from the Ministry of Education, Science and Culture of Japan to H. M. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The abbreviations used are: QP-S, ubiquinone binding protein that accepts electrons directly from soluble succinate dehydrogenase; QP-C, ubiquinone binding protein that occurs in the cytochrome bcl region; QP-N, ubiquinone binding protein inthe NADH-dehydrogenase segment; HPLC, high-performance liquid chromatography.
Portions of this paper (including “Materials and Methods,” part of “Results,” Tables 11-X, and Figs. 4-6) are presented in miniprint at 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 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-1322, cite the authors, and include a check or money order for $6.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal thatis available from Waverly Press.
337
Sequence of QP-C 30 5 10 25 15 20 Ac-Ala-Gly-Arq-Pro-Ala-Val-Ser-Ala-Ser-Ser-~g-Trp-Leu-Glu-Gly-Ile-Arg-Lys-Trp-Tyr-Tyr-Asn-Ala-Ala-Gly-Phe-Asn-Lys-Leu-Gly-
e
S
-
1
”
35 40 45 50 55 Leu-Met-Arg-Asp-Asp-Thr-Ile-His-Glu-Asn-Asp-Asp-Val-Lys-Glu-Ala-Ile-Arq-Arq-Leu-Pro-Glu-Asn-Leu-Tyr-Asp-Asp-Arg-Val-Phe___(
” “ , “ t + ” ” “ “ ”
4
’
“_I
+’
5-2
“ 7
-N-2-T-2” 7
--_I
CN-2-T-17 ” ” ” ” ” T ” -” ” ” ”
“
60
KN-2-T-
1 7 s - 3 -
c“-----s-4”-------(
t
s-5
65 70 75 80 85 90 Arq-I~e-Lys-Arq-A~a-Leu-Asp-Leu-Ser-Met-Arg-Gln-Gln-Ile-Leu-Pro-Lys-Glu-Gln-Trp-Thr-Lys-Tyr-Glu-Glu-Asp-Lys-Ser-Tyr-LeuN-3 “+” ” ” ” , ” , 4 ” ” 3+ +CN-2-T-4+ “” 2-T-51 1 C 3 N € c“--cN-3-C-2” ” 4 I CN-3-C-1’
2
” ” I
95 100 105 110 G~u-PrO-Tyr-Leu-Lys-Glu-Val-Ile-Arg-Glu-Arq-Lys-Glu-Arq-Glu-Glu-Trp-Ala-Lys-Lys.
_.. ., ””””C”””,‘
-S- 8
N-3-12-3 1
” ” 7 7 ”
” s - 9 -
c-s-10-
dN-3-C-4“I -!3-11+ -s-12+
” ” 7 ” ” ”
FIG. 1. Sequence study of succinylated QP-C. Arrows, +, -, and show solid-phase automated Edman degradation, manual Edman degradation, and carboxypeptidase digestion, respectively. The dotted arrows indicate ambiguous identifications due to low recovery of PTH-derivatives. 7,
chemical nature of the latteris available at present. Moreover, caution must be taken when comparing molecular weights determined by gel electrophoretic methods since the results vary according to the composition of the gel used and from one laboratory to another or even according to the person performing the experiment(10). Even if the polypeptides are distinct from each other, they should be constituents of the cytochrome bcl complex. QP-C is thought to bind with ubiquinone near thebenzoquinone ring to stabilize the semiquinone,whereasthe reactive groups of photolabeling compounds are situatedat the endof the side chain. The nature of ubiquinone binding with QP-C is uncertain, Because QP-C binds with the very hydrophobic ubiquinone, this binding maypossiblyoccur through the hydrophobic interaction indicated by spin immobilization (11). However, hydrophilic as shown by the present study, QP-Ca relatively is protein and the hydrophobic amino acidresidues arenot situated very close together. Only two segments, the N-terminal region and that near residue 20, are relatively hydrophobic. The possibility cannot be excluded that other areas also contribute to bindingwith ubiquinone by specific folding that generatesa hydrophobic patch. The secondary structure of QP-C was predicted by the method of Chou and Fasman (12) witha computer program. Fig. 2 shows the predicted location of the a-helix, @-sheet, @-turn conformation, and amino acid residues on helical wheels. The helical content was estimated to be about50% and that of P-sheet less than 10%.Thus, this proteinmay be classified as an all-a ora + p protein (13). As evident from the figure, charged amino acid residues are located on one side of the first,second, and fourth predicted helices and hydrophobic residues on the othersides. This may enable the formation of a specific region for interaction with hydrophobic cofactors such as coenzyme Q.Of course, these predictions are based on a method established by reference to soluble proteins and are not conclusive; but
they may facilitate an understanding of the structure of QPC. The amino acidsequence of QP-C was found to contain21 acidic residues (8 Asp and 13 Glu) and 25 basic residues (11 Lys, 1 His, and13 Arg). The isoelectric point of QP-C is then about 10, although it has been reported to be 3.6 (9). The reason for this discrepancy may be due possibly to the fact that the isoelectric point was determined for QP-C micelles with a weakly acidic detergent or that isolated QP-C may aggregate or form a complex with highly anionic compounds. As a whole, QP-Ccan be divided intothree regions: a relatively hydrophobic and basic N-terminal region (1-33), a middle region rich in aspartic acid (34-76), and a glutamic acid and lysine-rich C-terminal region (77-110). The C-terminal region has a particularly high content of hydrophilic amino acids and the charged amino acids are aligned in a row. This region may play an important role in the interaction with other proteins. The second ubiquinone-binding protein, QP-S,was isolated and some of its characteristics were determined (6, 14). Its amino acid composition is shown in TableI and suggests that these two ubiquinone-binding proteins are distinctmolecules. A structural analysis of QP-S is being carried out a t our laboratory and a comparison of these structures should provide important information on the binding of ubiquinone. In the bovine heart mitochondrial electron transfer chain, only two of the polypeptides so far sequenced have acetylated N termini, ie. subunit VI1 of cytochrome c oxidase (16, 17) and cytochromec (18,19). QP-C is thusa third example. The C-terminal residue of QP-C is lysine and it should be noted that some other components of the mitochondrial electron transfer chain possess the same residue: cytochrome c1 and two other small polypeptides in the cytochrome bcl complex (20-23) and subunits IV and VIIIa of cytochrome c oxidase (24, 25).
Sequence of QP-C
339
44
BO
'0 14
FIG. 2. Prediction of the secondary structures of QP-C. a-Helix, 8-sheet, andp-turn are shown by*, A, and [, respectively. Each helical region is also plotted as a helical wheel to show that the hydrophobic amino acid residues (0)are located on one side of the helix and the charged residues (9or @) on the other.
TABLE I Amino acid comDosition of ubiquinone binding proteins
20 -
10 Yeast14kDa BOvlne QP-C
QP-C 24-h hvdrolvsate ~.. ~
nal B l o c k i n g Group - T h eN - t e r m l n a lp e p t i d e d l g e l to fc i t r a c o n y l a t e d QP-C i n 14 % y i e l d . I t s a m n o acldCOrnPoIltlOn l i t h e i d m e a s P e p t i d e TT-1 1 5 f o l l o w s ;G l y I . O l ( 1 ) ~ A l a0 . 9 8 ( 1 ) , Arg 1 . 0 1 ( 1 ) . It gave no PTH-amino d r l d upon mdunal E b n md e g r a d a t l mb o t hb e f o r e and a f t e r a c i d t r e a t m e n t t o rem% t h e C i t r l c o n y l groups. Then t h l rp e p t i d e was a p p l i e dt ot h e m i i i p e c t r o m e t r yt o glVe t h et o t a l m13 O f 344. T h l s m a 5 1 c o r r e s p o n d e dt ot h e sum o ft h em l e c u l a rw e i g h t s O f component lnlno d c l d l group. The C - t e r m i n a lr e r l d w was a I I u n e dt o be a r g l n i n e j u d g e df r o mt h e a n da c e t y l specificity o f t r y p s i n a n d t h e r e s u l t 6 O f c a r b o x y p e p t i d a s e d i g e s t i o n O f P e p t l d e1 1 - 1 T h e r e f o r e . we s y n t h e s i z e dt w oa c e t y l a t e dt r l p e p t i d e i ,A c - G l y - A l a - A r ga n dA C - A l a - G l y - A r g , *hose p u r i t y was checkedby mars Spectrometry, HPLC andamino a c i d d n a l y s i l a f t e r a c i d h y d r o l y s i s . and compared t h e i r c h m m t o g r a p h l c r m b l l i t l e l on HPLC t o t h a t o f p u r l f i e d NThe n a t l vPee p t i d e was m - e l u t e d w i t h synthetic p e p t l d eA c - A l a t e m l n & l peptide (Fig. 6). From Glv-Ara on HPLC. w h l l e Ac-61"-Ala-Aro w a s e l u t e d imt b e f o r e t hnea t l vUee Y t l d e .
Table 11.
limine acld COmpOSLt10"S
CN-2
CN-1 ASP
s t a p h y l o c o c c a l Protease D i q e r t l o n a n d S e p a r a t i o n o f P e p t i d e s - T h e w c c i n y l a t e d QP-C (30 m~ was i g e l t e w i t nQ D Stap y ococca p r o t e a s ei n 2 m1 o f 0.1 n NH HCO at 40°C oiernighdt. Thde di:e!t'uas refparatehd'on I T;y$a?I HY-4OF column (2 X I80 c8) the same c o n d i t i o n s 15 above. The p e p t i d ef r a c t i o n I was f u r t h e rp u r i f i r db y DE-52 c o l u m (1.5 X 31 c m ) chromatography v r l n g a l i n e a r c o n c e n t r a t i o n g r a d i e n t Of ammniumblcarbonatefrom 0.05 M t o 0.8 M. The o t h e rf r a c t i o n s were s e p a r a t e db yp a p e re l e c t r o p h o r e s i sa t pH 6.5 or 3.6.
2.12121
CN-3
QP-C
9.11191
1.05111
12 2
1.0011)
0.89111
2.52(31
0.81111
0.9511)
0.96111
3.0513)
11.7112)
5 16
1.97121
4
0.19
4
4.72151
1.7312)
1.03111
8
2.0211)
2.95(21
1.11(1)
0.68111
0.69111
0.82(11
2.71131
1.85121
6
3.37131
3.69141
2.8Si31
10
1.6112)
0.99111
2.88131
6
0.92111
1.00(11
1.75121
1.5612)
5.9917)
11
2.69131
6.19161
3.99(41
13
1.04i11
i8
Of CYanOgen
peptides of QP-C
bromide
1.00111
4.21141
4
2
2 1
0.97(1)
-
IdentlflcatlonofN-terminalBlockinq Group C i t r a c a n y l a t e d (331 QP-C p r e p a r e db yt h e s m i l a r method t o r u c c i n y l a t i o n w a s d i g e s t e d w i t h t r y p s i n t r e a t e d w i t h I - t ~ l y l a m i d e - Z - p h e n y I e t h y l - c h l o m m t h y lk e t o n e( T P C K - t r y p s i n .e n z y m - s u b s t r a t er a t i o Of 1/50 (u/u)) a t room tempera t u r eo v e r n i g h ta n dt h ed i g e s t was s e p a r a t e db y gel f i l t r a t i o n on a ToyopearlW-4OF column a s a b o v ea n dP a p e re l e c t r O P h o r e l i sa t pH 3 . 6 . The p e p t i d en e g a t i v ei nn i n h y d r i n reaction and p o s i t i v ei nS d k a g u c h ir e a c t l o n ( 3 4 ) was r e c o v e r e da n da n a l y z e db yf i e l d desorption ?ass I p e c t r o l n e t r yw l t h JEOL JPIS-0300 doublefocusing m r r I p e c t o m t e r (35). Then t w op e p t i d e s Ac-Gly-Ala-ArgandAc-Ala-Gly-Arg, *ere s y n t h e s i z e d f r o m f-butyloxycarbonyl-am in^ a c i d s ( a b ) andtheelutionpmfilel O f t h e r ep e p t i d e s on HPLC were comparedWiththat O f t h ep e p t i d e d e r i v e df r o m OP-c. HPLC was c a r r i e do u t an an 00s column ( C o r m r i l C-18, 4 Y 150 c m ) u s i n g 0.05 % t r i f l m r O a c e t i C a c i d a n d 2 % acetonitrile as a s o l v e n t .
(21
(21
Total Yleldial
4
32
38
40
6
40
65
tryptic peptides Of Peptlde CN-1 TT-1T - 2
TT-2
T-3
0 3
2.9713) 0.45111 1.01i11
RESULTS CyanogenBromide P e p t i d e s - Edman d e g r a d a t i o n o f n a t i v e QP-C gave no PTH-amino a c i d s andtheN-terminus was a r r u m e dt ob eb l o c k e d . QP-C was n o tS o l u b l ei nd e t e r g e n t - f r e e s o l v e n t ,a n di t sC a r b o x y l( C ) - t e r m i n a la m i n oa c i dc o u l dn o t be determinedbyenzymicmethod. The amino a c i d c o n p o s i t i o n o f QP-C i s shown i n Table1and f a i r l yw e l la g r e e dw i t ht h a t deducedfromthesequence. QP-C Contained tw r e r i d u e ro fm e t h i o n i n ea n dt h e r e f o r e , i t was t r e a t e dw i t h cyanogen bmmide. T h e e l u t i o np m f i l e I I shown ~n F i g . 4. The f i r s t s m l l peak was j u d g e dt ob et h eh l n g ep r o t e i n ( 2 1 ) from I t s amino a c i dc o n p o l i t l o n .w h i c hh a s no methionine and was contaminated i n QP-c p r e p a r a t i o n . Fmm thesecondpeak, a large p e p t i d e c o u l db er e c o v e l e d whore N-terminalsequence was Arg-Asp. The amino a c i dc o m p o s i t i o n O f thllpeptideIndicatedthat it H I S d e r i v e db yl n c o n p l e t ec l e a v a g e O f m t h i o n y l - a r g i m n e bond a tr e s i d u e s IO and 71 l r e r u l t i n o t shown). The t h i r d and f o u r t h peakscontainedPeptides CN-3 and CN-2, r e s p e c t i v e l y .w h i c h were p u r i f i e db y DE-52 column c h m m t o g r a p h y . The P e p t i d e CN-I was e l u t e d i n a l o wr e c o v e r y l o n g a f t e l t h e c o l u m volumebytheHashingwith 60 X n-
CN-1
2 2.07121
Nomenclature - CN- and 8- r e f e r t o t h e p e p t i d e s d e r i v e d b y c y a n o g e n b r o m i d e Cleavage a n ds t a p h y l o c o c c a pl r o t e a s ed i g e s t i o n r. e s p e c t i v e l y -. T and -C- r e f e rt ot h ep e p t i d e s d e r i v e db ys e c o n dd i g e s t i o no fc y a n o g e nb m r n i d ep e p t i d e sw i t ht r y p s i n and c h y m t l y p s i n . TT- r e f e r st ot h ep e p t i d e sO b t a i n e db yP r O l O n g e dd i g e s t i o n O f p e p t i d e CN-1-1-1 rerPect?vely. v l t ht r y p s i n . Al t h e valuer are expressed i n ml p e r ml Of p m t e i n or p e p t i d e .
1
1 2. 0. 06 1( 1112 1
1.0711)
0.98111
1
1
1.09111
4 1.71(21
1.98121
5 1
1.13111 0.98111 1.0111)
1 1.96i21
3
2 2.3112)
1.30111
1
22 . 1 3 1 2 )
0 0.9511)
was n o tn o r m a l l ye l u t e df r o mt h e gel f l l t r d t i o n C N - I ( I - 3 2 )- S i n c et h l lp e p t i d e Column. theTecovery w a s v e r y low. The N-terminus O f t h i sp e p t l d e was b l o c k e da n dt h e p e p t i d e was d l g e r t e dv l t h1 P C K - t r y p s i n( 1 / 8 0 , r / w la t 40'C f o r 2 h. Threepeptldes CN-1-T-1 t o 1 - 3 were r e p a r a t e db y HPLC. S i n c eP e p t i d e 1 - 1 contained t w or e s i d u e so fa r g i n l n e . i t was f u r t h e r d i g e s t e d w i t h TPCK-trypsin (1/12. w / w ) a t 40'C o v e r n i g h t a n d fragments TT-1 and 11-2 were o b t a i n e d b y HPLC. The amino a c i d c o m p o s i t i o n s O f t h e r e p e p t i d e fragments are shown l n 11-2 andT-2 Yere conpletelysequencedby mnual E d m nd e g r a d a t i o n . Table 111.Fragments Fragment 1 - 3 Y I I a n a l y z e db ys o l i d - p h a s es e q u e n c e ra f t e ra t t a c h e dt ot h ed m i n o p r o p y l glass t h m u g hh o m o r e r i n el a c t o n em e t h o d and a l r m r tw h a l e Sequence was determined 15 % h o w ~n T a b l e IV. l h e5 t hr e l l d u e was i d e n t i f i e dt o b ea s p a r t i ca c l d i n t h i ra n d l y l i i ,v h l l et h e copreSpondlng relldue o f t h e s t a p h y l o c o c c a l p m t e a l e p e p t l d e 8 - 2 was asparagine I I shown l a t e r . IVI and t h el o s s a f t e r t h i ss t e p( T a b l e The recoverles o f PTH-amino a c i d sd r o p p e dd r a s t i c a l l y O f s i d eC h a l n amide gWup was seemed t o occur t h r o u g h c y c l i c i m i d ef o r m a t i o n d u r l n g t h e . arboxypeptidase 8 r e l e a s e do n l ya r g l n i n e Cyano en b r o m i d ec l e a v a g ea n dp e p t i d er e p a r a t i o n C (96 f r o m TT-1 a t 40-C f o r I h in 0.1 M Tris-HC1buffer, pH 8.0, a tt h ee n r y n e - s u b s t r a t e any o t h e r r a t l o o f Ill (ulu) and successive digestion u l t h c a r b o x y p e p t i d a r e A d l d n o t r e l e a s e
1.01(11
Ill 3
8
6
17
28 1 7
69
%s
amino
aclds.
1 2 3 4 8
SUC-Lys Trp Tyr Tyr Asp
6
nla
7 B
Ala
9 10
CIS Phe nsP Asn
11 12 13
I4 15
Suc-Lys
Le" Gly Le" Hse
3
0.84111
26
18 31 30 4. 3 2.2 2.6 4.1 2.2
0.7 0.5 0.6
1.1 1.7
1.1 0.6
111 18
2 32
342
Sequence of QP-C Table Y. Solld-phase Sequence d n a l y s ~ sof peptlde CN-2 cvcle
Table "11. Solid-phase Sequence analysls of peptlde CN-2-T-5
PTH-A.A. n m O l IHPLCI
cycle
37 64 68
I 2 3
8.4
5
16 26 20 7.0 5.1 17 17 7.2
9
10 11 12 13 I4 15 16 17 18 19 20 21
68 54 63 42 3.7 26
&la Leu Asp Leu ser Hse
3
4
6 7 8
PTH-A.A. m o l IHPLC)
1
2 4 5
6
Sequenced once using 86 n r m l of peptlde
8.4
6.6 'Table "111. Solid-phase sequence analysis of peptlde CN-3
5.0
5.7 7.2 8.0
cycle
PTH-A.A. n m l IHPLCI
5.5 3.9
1 2
3.4
22 23 24 25 26 27 28 29 30 31 32
1.5 1.4 1.3
3
2.0
4
1.0
5
1.8
0.9
6 7 8
1.1
9
3.4 3.1
Gin
0.7
0.9 0.8
6.9
10
Sequenced once u s i n g 96 m a l of peptlde
Glu Gln
1.5
lie Le"
7.4
7.4
Pro SuC-Lys 4.8 Glu GI" Gln Trp Thr SuC-Lys Tyr
11 12 13 14 15
0.4
33
-
Airg Glu
4.9
2.3 2.3 0.4 1.8 1.1 1.5
1.9
Glu Giu
1.8
1.1
Sequenced Once using 150 n r m l Of peptlde
Table V I .
Amino
acld COmpOlltlOtlS Of r r y p r x peptldes of Peptlde CN-2
T-1 T-3 T-2 Asp Thr
5.23151 1.03111
T-5
T-4
0.81111 0.6811)
HSe
Glu
0.91111
1
GlY Ala Val Ile Leu
His
nrg
3
1.1911)
0.95111 1.09111 1.87121
0.84111
2 2 3
1.99121
4
0.98111
2.06(21
1
0.74111
1.90121
1.04(11
0.98111
2
1.02111
6
3
3
6
37
63
52
50
P-6
P-6
Catlo"
P-6 PE6.5 PE3.6
PE6.5 0.25 -0.49
0.42 -0.06
0.59 0.11
0.72 0.59
Table X.
P-6
2.61131 1.02111
0.39 0.12 0.87111 0.92111
5.45151
0.30
0.14 1.05111 0.61111 0.91111
0.95111
0
0.81111 1.02i11 0.95111
lPE3.61 lPE6.51
3
3
1.05111
2.01121
2.05121
1.21111 0.96111
1.77(21 2.1112) 7
0.94111
0.16111
3.00(31
0
4
111
Total
13
10
10
14
3
~leld181
33
5
21
25
29
P-6
P-6
P-6
P-6
0.32 0.28 -0.18
%YS
-0.08
P-6 PE6.5
PE6.5
0.00 -0.82
0.32 -0.42
IPE3.61 0.20 1PE6.51 -0.68
Amino acld compasltlons of etaphylocaccal protease peptldes of QP-C
5-3
S-5 S-7 5-6
5-4
4.29141
S-8
1.10111
0.35
5-95-12 S-11 S-10 0.18
0.83111
1.02111
w
Rrg
rn mtal Yleld(81 Rnl Ulrlfl-
1.96i21
0.801ll
1.88121
1ll
1.001111.92121 Ill Ill
14 6
"
65 1532 VI1
25 I1 VI1
PE3.6 PE3.6 PE3.6
CarlLln 10.41
(0.4)
2.14(21 3.12131 1.02(11
7 51
I E-52
(0.01
111
12
11 15
I11
I
I11
0.98111
1.01111 111
11 34 I
E-52 1-0.51
1-0.11
PE6.5 1-0.91
7 1 71 5
PE6.5 PE6.5 DE-52 PE6.5 10.41
PE6.5 10.11
1.08(11
Ill
15
1-0.1)
1
1 2
1.29111 1.02111 1.21111 1.18111
H15
12 2
0.99111
carlo"
0.18 -0.25
1 1
0.26 0.951110.85111 Glu 0.99(11 0.16 3.29131 3.07131 2.09121 0.92111 1.04(1) 1.99121 PIC 0.85111 1.07(11 1.13111 1.21111 2.95131 GlY 1.20111 0.15 0.22 0.37 &la 2.68131 2.01121 0.90111 0.93111 0.15 Val 1.15111 1.21(1) 1.23111 0.63111 0.89111 Met 0.96111 1.61121 Ile 0.94111 0.90111 1.02111 0.50111 Ieu 0.9511) 0.98i11 2.06121 0.25 1.91121 0.16 1.04111 2.20121 1.91121 2.02121 0.95111 0.92111 1.75121 P k 1.01111 0.92111 0.16 1.99121 0.79111 0.98111 1.80121 0.98111 0.94111 0.93111 LYS
PE6.5
CN-3
0
Purifl-
4.04141 3.08131 0.86111
A?? Sar
GlY Ala va1 Ile Leu
I11 TCP
38
S-1 S-2 S-1'
mr
0.28 0.75111 4.06141 3.26131
~ r g (11
10
Purlf1- P-6
C-4
HLS
2 16
Yleldl81 72
Total
C-3
0.90111
ser 1 Glu 4.05(41 pro0.93(111.08111
TyT Phe Lys
0.96111
..r
Lys
1
0
0.91111 0.95111 1.94121
C-2
1.OOl11
0.22
ASP ThT
1
1.31111
TYr Phe Lys
1
2.35121
PZO
C-1'C-1
CN-3
1
ser
1
9
1,17111
3.3313)
Table I X . Amino acld COmpOSltlOnS Of ChymOtryptlC peptides Of Peptide
CN-2
HPIL
4
3
3
4
17
14
34
111 I1 PE6.5 PE6.5 PE6.5 PE6.5 10.11 1-0.31 HPIK
11
(-0.31
IV
(-0.51
40
Sequence of QP-C
343
c
a
Y
2.0.
