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THE SEQUENCES OF THE TRYPTIC PEPTIDES
FROM ACTINI:OIN
A thesis presented in partial fulfillment tor the degree of Doctor of Philosophy
at Maesey University.
Alan Carne December, 1 976
1 ABSTRACT
Actinidin is a plant thiol protease which is isolated froa the fruit of Actinidia chinensi�, the chinese gooseberry.
Determination of the primary amino acid sequence of actinidin was undertaken to extend the limited structural information
available on this group of enzymes, and therefore enable a better understanding of their physical and chemical properties.
The order of arrangement of the 220 amino acid residues in the primary sequence of actiB}din was determined from the sequences of the tryptic peptides. s-carboxy( 14 c2laethyl actinidin was dicested with trypsin, and the twelve tryptic peptides produced were initially separated into seven tractions by gel chromatography on Sephadex G-50. The first tour fractions contained tryptic peptides that were purified by DEAE-cellulose chromatography. The last three fractions contained peptides that were sufficiently small to enable purification by paper
techniques, and these peptides were sequenced directly by the dansyl-Edman method.
Further degradation of the tryptic peptides purified on DEAE-cellulose with either chyaotrypsin, thermolysin, pepsin or Staph1lococcus aureus v8 protease was necessary to provide
smaller peptides that could be sequenced by the dansyl-Edaan method. s . aureus VB protease was particularly useful in the
deteraination of aside residues, because of the enzyme speciticity for the carboxyl groups of glutamic acid.
The fourth tryptic peptide in the sequence of e.ctinidin could not be located in the tryptic peptide elution profiles of either the S ephadex G-50 or DEAE-cellulose columns. The sequence of this peptide was cieter.aiJled. froa a tr7ptic peptide obtained by
digestion of maleylated carboxymethyl actinidin.
The N-terminal of actinidin was determined by the dansyl Edman method, and the c-terminal by analysis of cyanogen bromide fragments, and by digestion with carboxypeptidase A.
Radioact ively labelling the ac t ive sit e cyst eine residue with iod o [ c2lacetic ac id , and subsequent purification of the 1 4
ii
radioact ive tryptic d igest peptid e , enabl ed the isolat ion of the t ryptic pept ide c ontaining the ac t ive sit e cysteine residue. Further digestion of this pept id e with chymotrypsin and
d e t erminat ion of the sequence of the smaller radioact iYe pept id e , provided t h e sequenc e about t he ac t ive sit e cysteine residue .
Alignment of t he trypt ic peptid e s t o reconstruc t t he primary sequenc e of ac tinidin was acc omplished wit h .information from cyanogen bromide fragment s , information from tryptic peptides o f maleylat ed carboxymet hyl actinidin , a nd information from the t hree dimensional X-ray crystall ographic struc ture of act inidin d etermined by Dr E.N. Baker.
The low proportion of basic residues a nd high proportion o f acidic residuea i n actinidin are i n agreement with t h e enzyme being an acid ic protein. Calorimetric analysis of t he t ryptophan residue c ont ent , using 2 -nitrophenylsulphenyl chlorid e , c onfiraed t he presence of six tryptophan residues in t he sequence of
actinidin.
The aaino acid sequences about the seven cyst eine r esiduea and the singl e hist idine residue in ac tinidin were very siailar to the analogous sequenc es in papain and other plant t hiol proteaaea. FUr thermore, comparison of the primary eequenoe of aotinidin with t hat of papain, and the fragaenta of sequence available for other plant thiol proteases , indicat ed a
c onsiderable homology t hroughout t he sequences of these prot eins.
ACKNOWLEDGEMENTS
I wish t o t hank my supervisor Dr C . H. Moore for his c ontinued int erest in the proj ec t , and for the benefit of his experienc e in the form of helpful suggest ions , t hroughout t h e course of t his work.
The useful d iscussions with Dr G.G. Midwinter and Dr E. N. Baker during the pro j ec t are much
appreciat ed , as is the assistance of Mr P. R. Mac donald and Dr I . M. Morrison with proof reading ot the thesis.
I wish also t o acknowledge t he e nc ourageaent given by my parent s and Pauline during the c ourse of t his work.
iii
CHAPl'ER I
1 . 1 . 1 . 2 .
CHAP!'ER I I
Sect ion 2 . 1 . 2 . 1 . 1 . 2 . 1 . 2 . Section 2 . 2 .
2 . 2 . 1 . 2 . 2 . 2 . 2 . 2 . 3 . Section 2 . 3.
2 . 3 . 1 . 2.3 . 2 . 2 · 3· 3·
2 . 3 . 4 . Sec t ion 2 .4 .
2 . 4 . 1 . 2 . 4. 2 . S ec tion 2. ;.
2 . ;. 1 .
Abstract
Acknowledgements Contents
List of Figures List of Tables Abbreviations
INTRODUCTION
CONTENTS
Actinidin a nd it s relat ion t o other prot eas e s . Det erminat ion of a prot ein primary sequenc e .
MATERIALS AND METHODS
MATERIALS METHODS
Prot ein purification.
Enzyme assay .
Gel el ectrophoresis of purified ac tinid in.
C hemical mod ification reac t ions.
iv
Page i iii iv viii xi xiv
1 .
1 4.
1 6 .
1 6 . 1 7 . 1 8.
Reduc tion and S·carboxymethylation of act inidin. 1 8.
React ion o t cyanogen bromid e with act inid in. 1 9.
Reac t ion of maleic anhydrid e with actinidin. 1 9.
Enzyme digestion ot act inid in.
'l'rypein. 20.
C hymotrypsin , thera olysin and s. aureue v8 pro t eae e . 2 1 . Pepsin.
Carboxypeptidase A·
C olumn chromatography . Sephadex gel fil trati on .
DEAE-c ellulose c hroma t ography .
Peptid e puri fication by paper t echniques.
High voltage paper electrophoresis .
2 1 . 2 1 .
24 ..
CONTENTS ( c ontd. ) 2 . 5. 2 . Paper c hromat ography .
2 . 5. 3 . Peptid e stain ing on paper.
2 . 5. 3 . 1 . Ninhydrin-cadmium reagent . 2 . 5. 3 . 2 . Chlorine-tol idine rea gent .
2 . 5. 3. 3 . Specific amino ac id st aining on paper.
( a ) Ehrlich reagent for trypt ophan.
(b) Arginine r eagent .
( c ) Pauly reac t ion for histidine and tyrosine .
V
Page 25.
2 5.
2 6 . 2 6 . 2? .
2 . 5 · 4. Peptid e elution from paper . 2 8.
S ection 2 . 6. Amino acid a nalysis . 2 . 6 . 1.
2 . 6. 2 . S ect ion 2 . ? .
2 . ?. 1 . 2 . ?.2 . Section 2. 8.
2 . 8 . 1 . 2 . 8. 2 . 2 . 8. 3.
2 . 8 . 4 . section 2 . 9.
CHAPTER I I I
Section 3. 1 . S ection 3 . 2 . S ection 3 · 3 · Section 3 . 4 .
3 . 4. 1 . 3 . 4. 2 . J . 4 . 3 . 3 . 4 . 4.
3 · 4 . 5·
3 . 4 . 6.
Det ermination of t he amino a c id c omposit ion of peptid e and prot ein samples.
Determination of trypt ophan . Determination o f radioact ivity . Liquid scint illatioh spectrometry.
Radioautography . Pept ide sequenc ing.
28.
2 9 .
2 9 . 30.
Dansyl N-t erminal procedure for pept id es . 30.
Dansyl -Edman proc edure for pept ide sequencing. 31 .
Rapid Edman d egradation. 32 .
Det ermination o f amide groups . 33.
Det erminat ion of the ac tive sit e cyst eine residue in ac tinidin .
RESULTS
Purification of act inidin. 35.
Amino acid c omposition o f act inidin. 38 . Purificat ion of the tryptic pept id es of act inidin. 40.
The s equenc e s of the trypt ic peptides. 4? .
Peptid e T 1 • 49.
Peptid e T2 • 50.
Peptide 13• 51 .
Pept ide T4• 59·
Peptide T5• 6? .
Peptide T6• 6 8.
3 . 4 . 9.
3 . 4 . 1 0.
3 . 4 . 1 1 . 3 . 4 . 1 2 . S ec tion 3 . 5·
3· 5. 1 .
S ec t ion 3. 6.
CHAPI'ER IV
S ec tion 4. 1 . S ec tion 4. 2 . S ection 4 . 3.
Sec tion 4. 4.
Sect ion 4. 5.
C ONTENTS ( oontd. )
Pept ide T7 • Peptide T8•
Pept ide T9•
Pept ide T1 0•
Pept ide T 1 1 • Peptide T1 2 •
Det ermination of the C-t erminal region of actinidin.
Det ermination of the C-t erminal region of aetinidin from the cyanogen bromide fragment s . ConfirmAtion o f the C -t erminal region o f
a c tinidin with carboxypeptidase A.
Det ermination of the amino acid sequenc e about
Ti
Page 78.
86.
92 . 94.
95.
98.
99.
1 02 .
the active sit e cyst eine residue in act inidin. 1 03 .
DISCUSSION
The purification of ac tinidin.
The amino ac id c omposition of ac tinidin.
Purification of the trypt ic pept ides of ac tinidin.
Determinat ion of the amino acid sequenc e of each trypt ic pept ide froa actinidin.
Peptide '1'1 • Pept ide t2 • Pept ide T3 • Pept ide T4•
Peptide T5•
Pe ptide T6•
Peptide T7 • . Peptide T8•
Peptide T9•
Pept ide '1' 1 0•
Peptide T 1 1 • Pept ide 'l' 12 •
The c-terminal region ot act inidin.
1 04 . 1 04.
1 05.
1 07 . 1 08.
1 08.
1 09.
1 1 o.
1 1 3 . 1 1 3 . 1 1 6 . 1 1 8.
1 1 9.
1 20.
1 2 1 . 122.
1 22 .
Section 4. 6.
Sec tion 4. 7.
Sec tion 4 . 8.
C ONTENTS (contd . )
Alignment of t he t ryptic peptides of actinidin.
The degree of homology between t he act ive sit e amino acid sequenc es from actinidin and some other pla nt t hiol proteases.
The degr ee of homology between the primary amino acid sequenc e of actinidin and ot her plant t hiol prot eases.
REFERENCES
vii
Page
1 24.
1 2 8.
1 31 .
1 35.
LIST OF FIGURES
Proteolyti� en�yme groups,
1. 2. Amino acid �equences containing the essential cysteine in some thiol proteolytic enzymes.
1. 3. General active site structure ot the plant thiol protease enzymes.
DEAE-cellulose chromatography ot actinidin.
3. 2. Sephadex G-50 chromatography ot the tryptic peptides ot carboxymethyl actinidin.
3 . 3. Composite peptide maps ot the electrophoretically mobile tryptic peptides or carboxymethyl
actinidin.
Composite DEAE-cellulose chromatography ot the tryptic peptides of carboxymethyl actinidin.
Chymotrypsin digest peptide maps ot peptide T3•
Thermolysin digest peptide maps ot peptide T3•
Reconstruction ot peptide T3 from chymotrypsin and thermolysin digest peptides.
Determination ot the amide and agid residues in peptide T3ChTa by the Offord mobility procedure.
3. 9. Sephadex G-75 chromatogra.phy of the tryptic
viii
Page 1.
4.
41,42.
45,46.
53.
55·
56.
57,58.
peptide& ot maleylated carboxymethyl actinidin. 60.
3. 11.
3. 12.
Maleyl diagonal peptide map at pH 6. 5 ot a chymotrypsin digest of the maleyl tryptic peptide containing peptide T4.
Reconstruction of the maleyl tryptic peptide obtained from fraction A of the Sephadex G-75 elution profile from chymotrypsin and s. aureus v8 protease digest peptide& to provide the sequence ot peptide T4•
Chymotrypsin digest peptide maps of peptide T6•
61.
66.
70.
Figure
3.14.
3.16.
3.18.
4.1.
LIST OF FIGURES (contd.)
Sephadex G-25 chromatography of peptides
obtained by digestion of peptide T6 with s. s�reus v8 protease.
Composite peptide maps of the chymotrypsin digest peptiqes from peptides GEa and GEb from peptide T6•
Reconstruction of peptide T6 from chymotrypsin and s . aureus v8 protease dig·est peptides.
Determination of the amide and acid residues in peptide T6GEbChTa2 by the Offord mobility
procedure.
Chymotrypsin digest peptide maps ot peptide T7•
Sephadex G-25 chromatography of peptides obtained by digestion of peptide T7 with s. aureus v8 protease.
Composite peptide maps of s. aureus v8 protease and pepsin di��st peptides from peptid� T7•
Reconstruction of peptide T7 from chymot.rypsin, pepsin �nd s. aureue v8 protease digest peptides.
Chymotrypsin digest peptide maps of peptide T8 • Sephadex G-25 chromatography of peptidea
obtained by digestion of peptide Ta wtth s. gureus v8 protease.
Reconstruction of peptide Ta from chymotrypsin, pepsin and s. aureus v8 prote�se digest peptides.
Thermolysin digest peptide maps of peptide T11•
Sephadex G-50 chromatogr9phy of the cyanogen bromide fragments from carboxymethyl actinidin.
Sephadex G-25 chromatography of the cyanogen bromide Sephadex G-50 fraction CB-II+III.
Alignment of the tryptic peptides of actinidin.
ix
71.
74.
75·
8 1.
8 4.
91.
97·
100.
100.
127.
Figure 4.2.
LIST OF FIGURES (contd. )
Amino acid sequences containi�g the essential cysteine in some thiol proteolytic enzymes.
4 . 3 . Amino acid sequences about the histidine
4.4.
residue in the active site of some plant thiol proteases.
Homologies among the available sequences of some plant thiol proteolytic enzymes.
X
Page
1 28 .
1 30.
1 32 , 1 33 .
LIST OF TABLES
Table
3 . 1 . Purification of ac tinid in.
Amino acid composition of act inidin.
3 . 3 . Peptide isolation data and amino ac id composition of pept ide T1 •
3 . 4 . Peptide isolat ion data and amino ac id c omposition of pept id e T2 •
3· 8 •.
Amino acid compos it ion of peptid e T3•
Peptide isolation data and amino acid composit ions of the c hymotrypsin digest peptides of peptid e T3 • Peptid e isolat ion data and amino ac id composit ions of the thermolysin digest peptid es from peptid e T3 • Peptid e isolation data and amino acid composit ions
of the chymotrypsin digest peptid e s from t he maleyl tryptic pept id e containing pept ide T4•
3 . 9. The peptid e isolation data and amino ac id
3. 1 0 .
3.1 1 .
3 . 1 2 .
compositions of the s . aureus V8 prot eas e digest pept id es of �ipt id e C hTA t hat was obtained by c hymotrypsin d igest ion of t he maleyl trypt ic pept ide containing pept id e
T4•
Amino acid composition of pept id e GE/Y obta ined by s. aureue VB prot e8s e digestion ot the maleyl t ryptic pept id e c ont aining pept id e T4•
Pept id e isolation data and amino acid composition of pept id e T5•
Amino ac id composit ion of peptide T6•
Peptid e isolation dat a and the amino acid
compositions of t he chymot rypsin digest peptid es froa pept ide T6•
Amino acid compositions ot trac tions GEa and GEb obtained by s. aureus v8 protease digest ion of peptide T6•
xi
Page
35.
39.
49.
50.
51 .
52·
62;
64.
67 . 68.
72 .
Tabl e
3.26.
LIST OF TABLES ( c ontd . )
Peptid e isolation data and amino acid c ompositions of t he chymotryptic peptid es froa peptid es GEs and GEb t hat were obtained by s . aureus v8 protease
.xii
Page
d igestion of peptide T6• 73.
Aaino acid composition of peptide r7• 78 . Peptid e isolat ion data and amino scid compositions
of the chymotrypsin digest peptide& froa peptide T7• 79.
Peptide isolation data and amino ac id c ompositions of the pepsin d igest pept ide s of peptid e GE1 t hat was obtsined by s. aureus
v8
protease d igestionof peptide T7• _82.
Pept ide isolation data and amino acid c ompositions of pept ides GE2 and GE3 t hat were obtained by
d igestion of pept ide T7 with s. aureus v8 prot ease . 83.
Aaino acid c omposition of peptid e T8• 86.
Peptid e isolation data and amino acid composit ions
of the chymotrypsin d igest peptide& from peptid e T8 • 87.
Pept ide isolation data and amino acid compositions of the pepsin digest pept ides from peptide GE1 t hat was obtained f�om peptid e Ta by digestion with
S. !Ureus V8 prot ease . 90.
Pept id e isolation data a nd amino acid c omposition ot peptide GE2 that was obtained from peptid e T8
by d igestion with s. aureue vS prot ease. 90.
Peptid e isolation data and amino acid
c oaposition of peptide T9• 92 .
Pept id e isolation data and amino acid compositions
of the chymotrypsin d igest peptides from peptid e T9• 93.
Peptide isolation data and amino acid coaposition of peptid e T10•
Pept ide isolation data and amino acid composition of peptid e T1 1 •
94.
xiii
LIST OF TABLES (c o ntd . )
Table Page
Pept id e isolation data and amino acid composit ions
of the thermolysin digest pept ides from pept id e T1 1 • 96.
Peptid e isolation data and amino ac id
c ompo sit ion of pept id e T1 2 • 98.
Peptid e isolation data and amino acid compositions of cyanogen bromide fragment s from ac tinidin.
Peptide isolation data and amino ac id c ompositions of the c hymotrypsin digest peptides from cyanogen bromid e fragment C B-IIa2 •
4. 1 . Amino ac id composition of some plant thiol prot eases.
4. 2 . C omparison of the amino acid c omposition of
ac tinidin with the amino acid compositions of the tryptic pept id es as det ermined from the amino acid sequence of each peptid e .
99.
1 01 .
1 06.
1 26.
BAW BAWP
N-�-CBZ-lys-j!NP Dansyl, DNS
DEAE DTT E. U . FDNB NpS
%
PITC PO POP PPO
S. aureus TFA
TPCK Tris
xiv
ABBREVIAT IONS
But anol/glacial ac e t ic acid/ dist illed water .
Butanol/glac ial acetic acid/dist illed water/pyridine.
N-�-carbobenzoxy-L-lysine-R-nitrophenyl ester.
1 -dimethylaminonaphthalene-5-sulphonyl Symbol used t o indicate direct dansyl -Edma n sequenc ing.
Diethylaminoethyl.
Dithiothreitol.
Enzrme unit (�mol substrat e transformed. min-1 ) . 1 -tluoro-2 , 4-dinitrobenzene.
2 -nitrophenylsulphenyl .
All percent a ges are weight/volume ( w/v ) unless otherwis e stated.
Phenylisothiocyanate.
1 , 4-di[2 - ( 5-phenyloxa zolyl )]benzene.
2 , 5-diphenyloxazole.
Staphylococcus aureus . Tri!luoroacet ic ac id.
L•(1 -t osylamido-2 -phenyl ) ethylc hl oromethyl ketone.
rria ( hydroxymethyl ) amino methane.
Amino acid abbreviations :
Ala Alanine
Arg Arginine
Asn Asparagine
Asp Aspartic acid
Asx Aspartyl or Asparagi nyl
CMCya Carboxymethyl cysteine
Cya Cysteine
CyS03H Cysteic a c id
Gln Glutamine
Glu Glutamic acid
Glx Glut amyl or Glutalllilll'l
Gly Glyc ine
His Hse
{;;>
Il e Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
Peft ides T
C hT Th p GE a b N
obtained
ABBREVIATIONS ( contd. )
Hist idine Homoserine
Homoserine lac tone Isoleucine
Leucine Lysine Methionine Phenylalanine Proline
S erine Threonine Tryptophan Tyrosine Valine
b;t enzzmic di�estion :
Trypt ic
C hymotrypt ic Thermolyt ic pep�ic
s. aureus v8 prot eoly t ic
�eptide ac idic c harged at pH 6. 5.
?ept ide basic charged at pH 6. 5.
�ept ide neut ral at pH 6 . 5 Enzyme EC numbers used are acc ording t o :
Rec ommendat ions ( 1 972 ) o t I . U. P. A . C. and the International Union o f Biochemistry.
Elaevier Scientific Publishing C ompany , Holland ( 1975 ) And Suppl ement I ( c orrections and additions (1975)) published in Bioc him. Biofhya. Acta ,
�.
1-45 ( 1976 ) .General abbrevia t ions :
XV
Are used acc ording to the " Policy ot the Biochemical Journal"
Biochem. J. , 153, - 1 -21 ( 1 976 ) .
