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UBC Theses and Dissertations

Plant protein chemotaxonomy : I. Disc Electrophoresis of Lasthenia seed albumins and globulins : II.… Altosaar, Illimar 1974

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PLANT PROTEIN CHEMOTAXONOMY I. DISC ELECTROPHORESIS OF LASTHENIA SEED ALBUMINS AND GLOBULINS I I . PARTIAL CHARACTERIZATION AND SEQUENCE STUDIES OF SAMBUCUS FERREDOXIN by ILLIMAR ALTOSAAR B. Sc. (Hon.), M c G i l l U n i v e r s i t y , 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December 1974 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of BOTANY The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date December 23, 1974 r. ABSTRACT P r o t e i n s i n d i r e c t l y r e f l e c t , v i a messenger-RNA, the i n f o r -mation coded i n DNA, and are thus considered to be t e r t i a r y se-mantides. Since p r o t e i n s are amenable to comparative analyses, they can provide a chemical b a s i s f o r a molecular phylogeny. This d i s s e r t a t i o n r e p o r t s the a p p l i c a t i o n of two approaches to the study of plant r e l a t i o n s h i p s using p r o t e i n c h a r a c t e r s . The f i r s t was an e l e c t r o p h o r e t i c comparison of seed storage pro-t e i n s from a l l twenty taxa of the genus Lasthenia. Albumin and g l o b u l i n f r a c t i o n s were e x t r a c t e d from dormant achenes. Each p r o t e i n sample was f r a c t i o n a t e d by d i s c e l e c t r o p h o r e s i s i n b a s i c 7% polyacrylamide g e l s . Mean R v a l u e s , c o e f f i c i e n t s of v a r i a t i o n , and 95% confidence i n t e r v a l s were c a l c u l a t e d f o r both types of p r o t e i n bands. S i m i l a r i t y c o e f f i c i e n t s , c a l c u l a t e d from the d i s t r i b u t i o n of homologous bands, were used to produce dendro-grams. A f f i n i t i e s among the taxa d i f f e r from the c o n v e n t i o n a l taxonomy of the genus. The second approach i n v o l v e d the p u r i f i c a t i o n and c h a r a c t e r -i z a t i o n of an i r o n - s u l p h u r p r o t e i n from a higher p l a n t and com-p a r i s o n with f e r r e d o x i n from other s p e c i e s . F e r r e d o x i n was i s o -l a t e d from leaves of Sambucus raaemosa L. by the f o l l o w i n g pro-cedure: 1) homogenization i n b u f f e r e d 50% acetone-water, 2) ion-exchange chromatography on s e v e r a l columns of DEAE-c e l l u l o s e , and 3) f i n a l l y p u r i f i e d i n good y i e l d by g e l f i l -t r a t i o n . The UV and v i s i b l e spectrum showed maxima at 277, 331, 423, and 466 nm. The p r o t e i n s u s t a i n e d an i n i t i a l p h otoreduction rate of 86 umoles NADP per mg c h l o r o p h y l l per hour. i i The amino a c i d composition was found to be Lyss, H i s 2 , A r g i , A s x i i , T h r 5 , S e r 7 , G l x i 7 , P r o 6 , G l y 7 , A l a 6 _ 7 , Cysi,, V a l 8 , H e 5 , Leu7, T y r 3 , Phe2 , and Trp i . The molecule has a molecular weight of 10,700 and contains 2 atoms of i r o n . The amino-te r m i n a l sequence i s Ala-Thr and the c a r b o x y l - t e r m i n a l sequence i s Leu-Thr-Ala. These p r o p e r t i e s are d i s c u s s e d i n r e l a t i o n to those of other angiosperm f e r r e d o x i n s . Experiments were performed to i n v e s t i g a t e the f e a s i b i l i t y of sequencing t h i s f e r r edoxin. i i i TABLE OF CONTENTS Page PART I: DISC ELECTROPHORESIS OF LASTHENIA SEED ALBUMINS AND GLOBULINS 1 Chapter I. INTRODUCTION 2 I I . EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . 6 1. MATERIALS 6 2. METHODS 6 l i t . RESULTS 9 IV. DISCUSSION . 16 BIBLIOGRAPHY . . . . . . . . . . . . . . 22 PART I I : PARTIAL CHARACTERIZATION AND SEQUENCE STUDIES OF SAMBUCUS FERREDOXIN 24 Chapter I. INTRODUCTION 25 II. EXPERIMENTAL PROCEDURE 35 1. MATERIALS 35 A. Plant Material 35 B. Chemicals and Solvents 35 2. METHODS 37 A. Preparation of Column Chromatographic Media 37 (1) DEAE-Cellulose . . . . . . . 37 (2) Sephadex Gels 38 B. Preparation of Ferredoxin 38 C I ) Preparation of Buffers . . . . . . . . . 38 C 2 ) I n i t i a l T r i a l s 39 (3) P u r i f i c a t i o n of Sambueus Ferredoxin . . 40 a. Homogenization 40 b. DEAE-Cellulose Adsorption 40 c. DEAE-Cellulose Chromatography . . . 41 d. Sephadex G-75 Chromatography . . . . 41 e. Desalting and Ly o p h i l i z a t i o n . . . . 42 C. Determination of the Molar Extinction C o e f f i c i e n t 42 D. Assay of Sambueus Ferredoxin A c t i v i t y . . . 43 i v TABLE OF CONTENTS (cont'd) Chapter Page II. E. Polyacrylamide Disc Electrophoresis . . . . 44 F. SDS Electrophoresis 45 G. . Amino Acid Analysis . . 45 (1) Automatic Amino Acid Analysis . . . . . 45 (2) Determination of Tryptophan . . . . . . 47 H. Determination of Non-Haeme Iron 48 I. Protein Modification 48 ('!;) tT'r.ichfboroaoetic Acid Treatment . . . . 48 (2) Reduction and S-3-Aminoethylation . . . 49 J. Digestive Procedures with Thermolysin and Trypsin 50 (1) Digestion of TCA-Treated Ferredoxin with Thermolysin . . 50 (2) Tryptic Digestion of AECys-Ferredoxin. 51 K. Column chromatography of Enzyme Digests .. 52 L. High Voltage Paper Electrophoresis of Peptides 53 M. NH 2-Terminal and Sequence Determination .. 54 (1) Dansylation of Peptides 55 (2) Dansylation of Protein 56 (3) I d e n t i f i c a t i o n of Dansyl Amino Acids . 56 (l/JIrnEdman Degradation 57 a. Coupling with Phenylisothiocyanate (PITC) ~.^r.- 57 b. Cleavage of Phenylthiocarbamyl-Peptide of -Protein 58 c. Removal of Diphenylthiourea (DPTU) 58 I I I . RESULTS 60 1. CHARACTERS ZATlONoSTUDIES . . . . . . . . 60 A. Preparative Work 60 B. Spectral Properties 67 C. Electron-Transfer A c t i v i t y 6 7 D. Homogeneity 7 2 E. Amino Acid Composition 7 5 v TABLE OF CONTENTS (cont'd) Chapter Page I I I . F. M o l e c u l a r Weight 82 G. Iron Content 8 3 2. PRELIMINARY SEQUENCE STUDIES 86 IV. DISCUSSION . . . . . . 93 APPENDIX THE AMINO ACID SEQUENCE OF FERREDOXIN FROM THREE HIGHER PLANTS 109 BIBLIOGRAPHY . . . . . . . . . . 110 v i LIST OF TABLES Page PART I TABLE I. The Goldfield Genus Lasthenia 3 II. Collections of Lasthenia Used for Disc Electrophoresis . 7 III. Mean R Values of Homologous Bands of Albumins and GlBbulins from Lasthenia Achenes 10 PART II TABLE I. Yield of Ferredoxin from Sambueus and Comparison to Yields from Other Plants 64 II. Absorption Maxima -of Plant Ferredoxins 69 III. C r i t i c a l Absorbance Ratios of Plant Ferredoxins . 70 IV. Molar Extinction Coefficients of Plant Ferredoxins 71 V. Amino Acid Analyses of Desiccated Hydrolyzates of Sambueus Ferredoxin 76 VI. Amino Acid Analyses of Undesiccated Hydro-lyzates of Sambueus Ferredoxin . 77 VII. Amino Acid Composition of Sambueus Ferredoxin . . 78 VIII. Amino Acid Composition of Plant Ferredoxins . . . 79 IX. Iron Content of Sambueus Ferredoxin . 8 5 X. Amino Acid Composition of Thermolytic Peptides of TCA-Treated Ferredoxin 90 y i i LIST OF FIGURES Page PART I FIGURE 1. Dendrogram for Unweighted Pair-Grouping of Lasthenia Taxa Based on Albumin Data 13 2. Dendrogram for Unweighted Pair-Grouping of Lasthenia Taxa Based on Globulin Data 14 3. Dendrogram for Unweighted Pair-Grouping of Lasthenia Taxa Based on Combined Albumin and Globulin Data . . .15 PART II FIGURE 1. Recommended C l a s s i f i c a t i o n of Iron-Sulphur Proteins ..26 2. Evolutionary Development Pattern of Ferredoxin . . . .31 3. The Phylogenetic Tree of Ferredoxins 33 4. Sephadex G-75 Chromatography of Ferredoxin-Contain-ing Solution Previously Fractionated on DEAE-Cellulose . ..66 5. Absorption Spectrum of Native and Deteriorated Ferredoxin from Sambucus raeemosa 68 6. Sambucus Ferredoxin-Mediated Photoreduction of NADP 73 7. Electrophoretic Pattern of Sambucus Ferredoxin . . . .74 8. SDS Electrophoresis of Fluorescamine-Labeled Ferredoxin and Molecular Weight Standards on 10% Polyacrylamide Gels 84 9. E l u t i o n Pattern of Peptides from the Thermolytic Digest of TCA-Treated Ferredoxin Chromatographed on a Column of Sephadex G-25 Fine (1.4 x 97 cm) . . ..88 10. Elution Pattern of Peptides from the Tryptic Digest of Sambucus AECys-Ferredoxin Chromato-graphed on a Column of Sephadex G-25 Fine (1.4 x 97 cm) • .92 v i i i ABBREVIATIONS AECys Aminoethylcysteine AEI 3-(2-aminoethyl)indole CMCys Carboxymethylcysteine Dansyl l-dimethylaminonaphthalene-5-sulphonyl DEAE Dimethylaminoethyl NADP Nicotinamide adenine dinucleotide phosphate PITC Phenylisothiocyanate PTC Phenylthiocarbamyl TCA Tri c h l o r o a c e t i c acid TEMED N,N,N',N'-tetramethylethylenediamine TFA T r i f l u o r o a c e t i c acid TPCK L-(l-tosylamido-2-phenyl)ethyl chloromethyl ketone T r i s Tris(hydroxymethyl)amino methane i x 'See vai t e k i r i on piihendatud minu vanematele, Lainele ja Heinole, kelle armastuse ja ohutuse kaasabi on voimaldanud minu oppinguid loppule v i i a . This thesis is dedicated to my parents, Laine and Heino. Their love and encourage-ment have made my studies possible. x ACKNOWLEDGEMENTS I am v e r y t h a n k f u l to Dr. B.A. Bohm and Dr. I.E.P. T a y l o r f o r t h e g u i d a n c e and e n t h u s i a s t i c s u p p o r t w h i c h t h e y o f f e r e d me t h r o u g h o u t the c o u r s e of my s t u d i e s . I am e s p e c i a l l y g r a t e f u l t o my s u p e r v i s i n g committee members, D r s . B.A. Bohm, I.E.P. T a y l o r , J . Maze, and V.C. R u n e c k l e s , and to Dr. C . 0 . P a r k e s f o r t h e i r i n t e r e s t and k i n d n e s s shown to me. T h e i r c a r e f u l y e t prompt e d i t i n g was v e r y h e l p f u l and e n c o u r a g i n g d u r i n g t h e p r e p a r a t i o n of t h i s t h e s i s . I a l s o t h ank Dr. P a r k e s f o r many u s e f u l d i s c u s s i o n s r e g a r d i n g s e q u e n c e a n a l y s i s , and f o r making h i s l a b o r a t o r y f a c i l i t i e s f r e e l y a v a i l a b l e d u r i n g c e r t a i n p a r t s of t h e r e s e a r c h . I thank Dr. R. O r n d u f f f o r s u p p l y i n g t h e Lasthenia p l a n t m a t e r i a l and f o r h i s d i s c u s s i o n s of t h e seed p r o t e i n d a t a . B o t h Dr. G. D u p u i s and Dr. F. W. C o l l i n s o f f e r e d many h e l p f u l s u g g e s t i o n s r e g a r d i n g l e a f p r o t e i n e x t r a c t i o n . Dr. R. O l a f s o n s h a r e d much u s e f u l i n f o r m a t i o n c o n c e r n i n g p r o t e i n chem-i s t r y . Mr. S. Bor d e n p e r f o r m e d many computer a n a l y s e s w i t h e n t h u s i a s t i c i n t e r e s t . Mr. K. J e f f r i e s and Mr. M. D a v i e s c o n -s t r u c t e d new equipment w i l l i n g l y when i t was q u i c k l y needed. Mr. M. McLeod k i n d l y h e l p e d i n o b t a i n i n g c h e m i c a l s and m a t e r i a l s . To a l l t h e s e i n d i v i d u a l s and a l l o t h e r members of t h e d e p a r t m e n t who h e l p e d me i n ways l a r g e or s m a l l , I e x t e n d my h e a r t f e l t t h a n k s . Y i a n n a Lambr.ou, my w i f e , m e r i t s a v e r y s p e c i a l a c k n o w l e d g e -ment i n t h i s t h e s i s . By h e r c o m p a n i o n s h i p and c o n s t a n t f a i t h xi i n the undertaking, she i n s t i l l e d me with p a t i e n c e and s t r e n g t h . Although committed to pursuing her own graduate s t u d i e s , she ne v e r t h e l e s s devoted much time and concern to make my work that much more rewarding and meaningful. I am g r a t e f u l to the N a t i o n a l Research C o u n c i l of Canada and the U n i v e r s i t y of B r i t i s h Columbia f o r suppo r t i n g me with Postgraduate S c h o l a r s h i p s and Teaching A s s i s t a n t s h i p s , r e s p e c t i v e l y . This study was funded by grants to Drs. B.A. Bohm and I.E.P. T a y l o r from the N a t i o n a l Research C o u n c i l of Canada. x i i P A R T I DISC ELECTROPHORESIS OF LASTHENIA SEED ALBUMINS AND GLOBULINS 1 CHAPTER I INTRODUCTION Lasthenia (Compositae: t r i b e Helenieae) i s a small genus occurring i n western North America with a single species native to Chile CI) . In North America the genus shows most d i v e r s i t y i n C a l i f o r n i a , where i t contributes to the f l o r a l displays of early spring i n a conspicuous manner. It . a l s o ranges from northern Vancouver Island to northern Baja C a l i f o r n i a , the Channel Islands, Guadalupe Island, and inland to central A r i -zona. The sixteen species (which are i n six sections, Table I) are e c o l o g i c a l l y and biochemically diverse, occupy a wide range of habitats, and generally show s t r i k i n g i n t e r s p e c i f i c d i f -ferences i n many morphological and chromosomal c h a r a c t e r i s t i c s (1). The systematics of Lasthenia has been the subject of a recent monograph (1). More recently, the nature of t h e i r flavonoid constituents has been investigated (3, 4, 5, 6). Members of the genus produce four anthochlor pigments Ctwo aurones and two chalcones), two l u t e o l i n glycosides, eight, quercetin glycosides, one kaempferol glycoside, six p a t u l e t i n glycosides, and one patuletin bisulphate compound C4). In general, however, each species exhibits a d i s t i n c t i v e array of flavonoids, and within some species there are as many as three "flavonoid races" which d i f f e r i n t h e i r flavonoid constituents (6). . . Proteins, being t e r t i a r y semantophoretic molecules, inherently possess a greater p o t e n t i a l to r e f l e c t more accurate-l y evolutionary history than do episemantic molecules such as 2 TABLE I 3 THE GOLDFIELD GENUS LASTHENIA^ Section Taxon Baeria L. chrysostoma, (Fisch. & Mey.) Greene L . macrantha (Gray) Greene subsp. macrantha subsp. bakeri (J. T. Howell) Ornduff subsp. prisca Ornduff Burrielia L. debilis (Greene ex Gray) Ornduff L. leptalea (Gray) Ornduff L. microglossa (DC.) Greene Platycarpha L. platycarpha (Gray) Greene Lasthenia L. kunthxi (Less.) Hook & Arn. L . glaberrima DC. Hologymne L. chrysantha (Greene ex Gray) Greene L . glabrata Lindl. subsp. glabrata subsp. coulter1 (Gray) Ornduff L. ferrxsiae Ornduff Ptilomeris L. fremontix (Torr. ex Gray) Greene L. burkex (Greene) Greene L. conjugens Greene L. coronarxa (Nutt.) Ornduff L. minor (DC.) Ornduff subsp. minor subsp. marxtxma (Gray) Ornduff a: According to Ornduff (1, 2). 4 flavonoids (7). The advantages of using protein data, especi-all y those derived from electrophoretic techniques, in studying plant systematics have been discussed (.8, 9). In studies of Ag-vopyvon". and Hordeum, disc electrophoresis in polyacrylamide gels of the storage proteins from seeds has shown that the band patterns obtained are: (a) genetically determined and not mo-dified by environmental factors; (b) different enough that species and (in some cases) infraspecific or racial populations may be distinguished; and (c) sufficiently conservative as to ac-curately reflect the specific relationships within a genus (9). Studies with legume seed proteins have also shown that they are not formed by a random mechanism,, and are indeed tertiary semantides (10). Boulter, Thurman, and Derbyshire pointed out that during the past half century, the proteins of seeds have been classified on various bases, including functional, sol-u b i l i t y , and structural or cytological c r i t e r i a (10). A ge-nerally accepted division of seed proteins is that of albumins, globulins, prolamins., and glutelins. As proposed by Osborne, albumins are soluble in water and dilute salt solutions; globulins are soluble in salt solutions but are not, or only slightly, soluble in water; prolamins are soluble in 70-80% (w/v) ethanol but are not soluble in absolute ethanol nor in water; and glutelins are soluble in dilute acids and alka-l i s but not in water, salt solutions nor aqueous alcohol (11). It is now recognized that enzymes are found in the albumin fraction and that the globulin fraction contains the storage protein (10). 5 The albumin fractions of seeds from seventeen species of the Leguminoseae.have been fractionated by polyacrylamide gel disc electrophoresis and shown to be of taxonomic use-fulness (12). A similar but more extensive study of the legume seed globulins has shown that certain tribes possess distinctive protein band patterns (10). In the genus Cvo-talaria, the seed globulin patterns of 24 species have shown reasonable correlation with the existing sectional taxonomy of the genus (13). As part of a continuing chemosystematic survey of Lasthenia, an electrophoretic study of the albumin and globulin seed fractions of Lasthenia waseuhder.takeni.toeinves-tigate the taxonomic usefulness of these seed protein chara-cters in the genus. CHAPTER II EXPERIMENTAL PROCEDURE 1. MATERIALS Seed material was obtained from Dr. R. Ornduff of the Botany Department, University of C a l i f o r n i a at Berkeley. For each taxon, dormant achenes from several plants (see Table II for c o l l e c t i o n numbers) were used. N,N,N',N'-tetramethylethylenediamine (TEMED), N ,N' -methylenebisacrylamide, acrylamide, and r i b o f l a v i n were obtain-edt-fromc Eastman Kodak Company of Rochester, New York. A l l other chemicals were from l o c a l suppliers. 2. METHODS Achenes were separated from chaff and cleaned. Proteins were extracted using modifications of the method of Fox, Thur-man, and Boulter (12). Achenes (1 part w) were ground with 5% (w/v) K 2 S O 4 (10 parts v) i n a mortar and s t i r r e d for 20 min. The s l u r r y was centrifuged at 1,000 x g for 10 min at 4 C and the supernatant was dialyzed against 4 l i t r e s of d i s t i l l e d water for 24 hr. The dialyzate was centrifuged at 1,500 x g for 10 min at 4 C and the supernatant was l y o p h i l i z e d . The freeze-dried proteinuwasnrefefr.ed2tolasf the "alBurtiindf raetionf orTfte p r e c i p i t a t e wasrresuspehded0d!nxl2 ml-of 5% K 2 S O 4 , dialyzed for 24 hr and cen-tr i f u g e d at 1,500 x g . The p e l l e t was suspended i n d i s t i l l e d water and l y o p h i l i z e d (globulin f r a c t i o n ) . A l l work was done at 4 C. 6 7 TABLE II COLLECTIONS OF LASTHENIA USED FOR DISC ELECTROPHORESIS Taxon C o l l e c t i o n L . chrysostoma Near Dozier, Solano Co., 4941 L. macrantha subsp. macrantha Point Reyes, Marin Co., 4140C L. macrantha subsp. bakeri Garcia R., Mendocino Co., 4709 L. macrantha subsp. prisca Rogue R., Curry Co., Oregon, Chambers 2466 L . debilis Elderwood, Tulare Co., 4788 L. leptalea Atascadero, San Luis Obispo Co., 4976 L. microglossa Mt. Diablo, Contra Costa Co., 4807 L. platycarpha Byron, Contra Costa Co., 4718 L. kunth.li Coline, C h i l e , Schlegel 3974 I. glaberrima Valley Home, Stanislaus Co., 4726 L. chrysantha Hanford, Kings Co., 4784 L. glabxata subsp, glabrata Garrapata Cr., Monterey Co., 4983 L. glabrata subsp. coulteri Sorrento Slough, San Diego Co., Witham 200 L. ferrlsiae Soda Lake, San Luis Obispo Co., Twisselmann 438 2 L. fremontii Soda Lake, San Luis Obispo Co., Twisselmann 4450 L. burkei East Windsor, Sonoma Co., 6067 L. conjugens Imola State Hosp., Napa Co., 4129 L. coronaria U. C. Riverside, Vasek, s. n. L. minor subsp. minor Maricopa, Kern Co., Twisselmann 4377 L. minor subsp. maritima South Farallon Id., San Fran-cisco Co., 6130 a: Unless otherwise indicated, a l l c o l l e c t i o n s are those of Ornduff. Vouchers are at University of C a l i f o r n i a , Berkeley. L o c a l i t i e s , except where noted, are i n C a l i f o r n i a . 8 Protein concentration was determined by the method of Waddell (14). For each protein f r a c t i o n , 8 r e p l i c a t e samples (200 ug/0.1 ml of 10% (w/v) sucrose) were separated by disc electrophoresis on 7% polyacrylamide gels (pH 9.0) which were 6 cm long and prepared according to Davis (15). Gels were run at 4 ma per tube for 50 min at 4 C and then stained i n 1% amido black i n 7% acetic acid for 1 hr. Protein band mobility (R^) was expressed as a f r a c t i o n of the mobility of the bromo-phenol blue front-tracking dye. Distances were measured from the middle of each band. The mean R . value, c o e f f i c i e n t of v a r i a t i o n , and 95% confidence i n t e r v a l of bands appearing i n half or. more of the r e p l i c a t e gels were calculated with the aid of an IBM 1130 com-puter. The position of each mean with i t s confidence i n t e r v a l was proportionally plotted on 1-m long paper by a Calcomp 565 d i g i t a l incremental drum p l o t t e r , with the taxa l i s t e d side by side. On this, large scale i t was easier, to determine band homology. Bands were considered homologous i f t h e i r con-fidence i n t e r v a l s overlapped more than 50%. In order to com-pare protein bands and obtain an index of s i m i l a r i t y for each pair of taxa, the s i m i l a r i t y c o e f f i c i e n t (SI) of Jaccard (Sneath) was used: SI = IA/(IA + IBC), where IA = number of homologous bands present i n both taxa; and IBC = number of bands present i n only each one of the two taxa (16). The s i m i l a r i t y co-e f f i c i e n t s were used to c l u s t e r the taxa by the commonly used unweighted pair group method with average linkage (16). CHAPTER III RESULTS The average c o e f f i c i e n t s of v a r i a t i o n for a l l albumin and globulin bands which "appeared i n t h i s study were 0.028 and 0.044 respectively. While both valuesaar.ev^ithin 5% experimental error, the seed globulins may be less s u i t -able for use as electrophoretic characters than the seed albumins. Homology of bands for the albumin (61 bands) and globulin (20 bands) fractions was determined from computer calculated means-R^ values and by comparison of confidence' in t e r v a l s on machine plotted 31 fm, graphs. Homologous band groupings for both seed protein fractions are shown i n Table --III. Ea>sj;1mnj%a:. m&^<%g;V.ds:sxii' possessed the greatest number of albumin bands (13) while the least number of albumin bands (4) appeared i n Eaxsitfaemva;., g$afamrt.ar subsp. gfrcffizLct/txiu- Albumin band 8, the most common (R 0.135-0.145) occurred i n 11 of the taxa. P LastTfcehvammirioiB:- subsp. md'nv4£mu\ showed the most globulin bands 05) while both Ba^>t]^n<8.a\~ptiemdntiibi. and XBoe&Mceri-ba :• miar;b-rgZossu*- showed the fewest (1) . The most common globulin band OR^  0.3-0.314) occurred i n only s i x of the taxa. Each species has a unique protein band p r o f i l e . Dendrograms, ind i c a t i n g the degree of phenetic s i m i l a r i t y among the taxa, were produced for the albumin characters, the globu l i n characters, and for both protein characters combined (81 bands i n t o t a l ) . These are repre-sented i n Figures 1, 2, and 3 respectively. 9 TABLE I I I . MEAN R VALUES OF HOMOLOGOUS BANDS OF ALBUMINS AND GLOBULINS FROM LASTHENIA ACHENES P • . .. • H •a: ft; ' o ft; ty CO o H ft; =5 CO ft. p. &. ft. tn tn tn P9 o tn tn tn D •< t-< a * as CO E l o a: . % Co •=« >1 ft; ft; ft; ft; c j Co to as to S : S : ft; ft) ft; h co y to cq ^ ^ tn tn co ft. S : &j t a H to to ft; H & H - ^ ^ ^ H =^ t o t a k j - ^ H f t ; s : t i E H N E - i t a S ^ t j O H f t ; ' ^ ; - ^ ^ : to H to ^ • ^ t o H a j c a c o f t i f t ; H <o ft) ^ H E H ft; E i h c o N c o c o ft; S : ^ ; ^ to ft, to -J: &: ^ ft; •>( >u ft;. ta ft: =s c a t a N ^ ^ ^ c c ^ ^ t a f t i ^ t o Q ^ S ; ft,«cotoCocj C C P Q C O ft; o ft: 5; p. ft. tn tn pa CO U> ft; tn CO '. -_ ft; ft; O o O ft; ^ o H • . o S: S: >o. TAXA (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) ( 20 ) . ALBUMIN BAND NO. 1 0 . 0 6 4 0 . 0 6 7 0 . 0 6 8 0 . 0 6 3 2 0 . 0 7 8 0 . 0 7 7 0 . 0 8 2 0 .077 0 .079 3 0 . 0 9 7 0 . 0 9 3 0 . 0 9 0 0 .093 0 .091 0 .086 4 0 .102 0 . 1 0 5 5 0 . 1 1 7 0 . 1 1 8 6 0 . 1 2 4 7 0 .132 0 . 1 2 9 0 . 1 3 3 8 0 . 1 4 0 0 . 1 3 5 0 . 1 4 2 0 . 1 3 8 0 . 1 3 9 0 . 1 4 5 0 . 1 3 7 0.141 0 . 1 4 5 0 .145 0 . 1 4 3 9 0 .154 0 .151 10 0 .161 11 0 . 1 7 5 12 0 . 1 9 4 0 .192 0 .188 13 0 . 2 0 4 0 . 2 0 4 TABLE I I I . MEAN R VALUES OF HOMOLOGOUS BANDS OF ALBUMINS AND GLOBULINS FROM LASTHENIA ACHENES (CONT'D) P TAXA (1) (2) (3) (4) (5) (6) (7) (8) (9) .(10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) ALBUMIN BAND NO. 14 0 .212 0 .213 15 0 . 2 2 0 0 .219 0 . 2 1 9 16 0 . 2 3 3 17 0 .241 18 0 . 2 5 6 0 .253 0 . 2 5 9 0 .258 0 . 2 5 7 19 0 . 2 6 9 0 . 2 6 8 0 . 2 6 6 0 .263 0 .267 0 . 2 6 5 20 0 .272 21 0 . 2 8 0 0 .278 0 .276 0 .280 0 . 2 8 0 0 .282 22 0 .286 23 0 . 2 9 3 24 0 .301 i 0 . 3 0 0 25 0 . 3 1 3 0 . 3 1 4 0 .312 26 0 . 3 3 6 0 . 3 3 6 27 0 . 3 6 8 28 0 . 3 7 7 0 . 3 7 5 29 0 .384 30 0 . 4 1 5 0.411 0 . 4 1 0 0.411 6 . 407 0 . 4 1 4 31 0 . 4 1 9 0 .422 0 .418 0 . 4 2 5 32 0 . 4 3 9 0 . 4 4 0 0 . 4 3 6 0 .443 33 0 .452 0 . 4 5 4 0 .456 34 0 . 4 6 3 0 .462 0 . 4 6 0 0 . 4 6 5 35 0 .482 0 . 4 7 9 0 . 4 7 4 0 . 4 7 7 36 0 .501 0 .495 0 . 4 9 5 37 0 . 5 1 0 0 .504 0 .512 0 .507 0 . 5 0 8 0 .502 38 0 . 5 2 5 0 . 5 2 8 0 .525 0 . 5 2 0 39 0 . 5 3 9 0 . 5 4 3 0 .542 40 0 .551 0 . 5 5 8 0 .564 0 . 5 5 5 0 . 5 5 5 41 0 . 5 7 3 0 . 5 7 3 42 0 . 5 9 0 43 0 . 5 9 7 44 0 . 6 2 3 0 .614 0 .617 0 . 6 1 7 45 0 . 6 3 9 0 . 6 4 5 0 .638 0 . 6 2 9 0 . 6 3 4 4 6 0 . 6 5 9 47 0 . 6 7 8 TABLE I I I . MEAN R VALUES OF HOMOLOGOUS BANDS OF ALBUMINS AND GLOBULINS FROM LASTHENIA ACHENES (CONT'D) P TAXA (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) ALBUMIN BAND NO. 48 0 .683 0 . 6 8 7 49 0 .696 0 . 6 9 7 0 . 6 9 3 50 0 .700 0 .702 51 0 .706 0 . 7 0 8 0 . 7 1 3 0 .713 0 .712 0 . 7 1 0 52 . • • ' • . 0 . 7 3 3 53 0 .741 54 0 .752 0 . 7 5 5 55 0 . 7 6 7 56 0 . 7 8 3 57 • 0 .794 58 0 . 8 3 8 0 .836 0 . 8 3 0 59 0 . 8 8 7 0 . 8 8 9 0 . 8 9 0 60 0 .932 , 0 .929 0 . 9 2 9 61 0 . 9 5 6 GLOBULIN AND NO. 1 0 . 0 5 7 2 0.080 0 . 0 6 9 0 .076 0 .076 3 0 . 0 8 7 0 . 0 9 0 4 0 . 1 0 5 0 .098 5 0 .112 6 0 . 1 4 3 0 . 1 4 3 0 . 1 4 9 •'• . .• 0 .136 7 .. --n; i :• : 0.162 8 0 . 1 7 9 0 . 1 8 5 0 . 1 8 5 ' . 9 0 . 1 9 7 0 . 2 0 2 0 .192 ' 10 0 . 2 1 0 0 .208 0 .213 0.212 0 . 2 1 0 11 0 . 2 1 9 0 . 2 1 7 12 0 . 2 2 5 0 . 2 2 6 0 .225 0.226 13 0 .231 0 . 2 3 6 0 .231 14 0 . 2 4 8 0 .248 0 .251 15 0 . 2 6 9 0 . 2 6 7 16 0 . 2 8 0 0 . 2 8 5 17 0 .296 0 . 2 9 6 0 . 2 9 3 18 0 . 3 0 0 0 . 3 1 0 0 .314 0 .309 0 . 3 0 4 0 .301 19 0 .425 20 0 .473 L. chrysantha L. glaberrima L. debilis L. glabrata s u b s p . glabrata L. macrantha s u b s p . prisca L. kunthii I. platycarpha •L. minor 6 u b s p . maritima L. leptalea L. glabrata s u b s p . coultcrl L. macrantha s u b s p . macrantha L. m a c r a n t / i a s u b s p . bakcrl L. ferrisiao L. fremontii L. coronaria L. chrysostona L. minor s u b s p . minor L. microglossa L. conjugens L. burkei 0.0 0.1 0.2 0.3 0.4 0.5 CORRELATION COEFFICIENT F i g . 1 Dendrogram for unweighted pair-grouping of Lasthenia taxa based on albumin data. L. chrgsantha L. debilis L. minor subsp. minor L. glabrata subsp. coulter! L. ferrisiae L. chrysostoma L. conjugens L. burkei L. glabrata subsp. glabrata L . platycarpha L. coronaria L. minor subsp. maritima L. fremontii L. glaberrima L. macrantha subsp. prisca L. kunthii L. microglossa L. macrantha subsp. macrantha ' L . macrantha subsp. bakeri L. leptalea 0.0 0.2 0.4 0.6 CORRELATION COEFFICIENT 0.8 1.0 F i g . 2 Dendrogram for unweighted pair-grouping of Lasthenia taxa based on globulin data. - L . chrysantha -L. debilis -L. minor subsp. minor -L. microglossa -L. conjugens -L. burkei -L. platycarpha -L. glabrata subsp. coulteri -L. minor subsp. maritima -L. macrantha subsp. macrantha -L. macrantha subsp. bakeri -L. leptalea -L. ferrisiae -L. chrysostoma -L. coronaria -L. glaberrima -L. glabrata subsp. glabrata -L. macrantha subsp. prisca -L. kunthii -L. fremontii 0.0 0.1 0.2 0.3 0.4 CORRELATION COEFFICIENT 0.5 0.5 F i g . 3 • Dendrogram for unweighted pair-grouping of Lasthenia taxa based on combined -albumin and globulin data. CHAPTER IV DISCUSSION Lasthenia has been divided into six sections (Table i) on the basis of morphology, cytology, and hybridization studies CI)• The flavonoid chemistry of the genus supports t h i s sectional treatment (4). In the present.study, each protein f r a c t i o n resulted i n a very d i f f e r e n t c l u s t e r i n g of the taxa. For example, i n the albumin dendrogram (Fig. 1), 15 taxa clustered into six groups at an a r b i t r a r y c o r r e l a t i o n c o e f f i c i e n t of 0.2 or higher. Only four of these 15 taxa {Lasthenia macrantha subsp. prisca and Lasthenia kunthii, Lasthenia macrantha subsp. macrantha and Lasthenia macrantha subsp. bakeri) are clustered together at a comparable l e v e l i n the globulin dendrogram CFig. 2). Other such comparisons indicate equal disagreement between the two protein c l a s s i -f i c a t i o n s . This indicates the distinctiveness and independence of the two seed protein f r a c t i o n s , and t h i s must be considered when either of them i s used as an evolutionary indicator. The sectional groupings of the taxa were not c l e a r l y r e f l e c t e d i n any of the dendrograms. In the albumin dendrogram, only two of the six clusters mentioned above contained species belonging to the same section, and i n each.of these a t h i r d species belonging to another section also was included. In the globulin dendrogram, a l l four taxa of. section Hologymne clustered together i n a 9-member group at a low c o r r e l a t i o n c o e f f i c i e n t of 0.118. Due to the influence of the d i s s i m i -l a r i t y of t h e i r albumins, this.section.was further dispersed 17 i n the other two dendrograms. Section Baeria contains two species, Lasthenia chry sostoma and Lasthenia macrantha. The l a t t e r has been subdivided into three subspecies on morphological and cyto-l o g i c a l grounds (1, 2). Lasthenia macrantha subsp. macrantha Cn =24) has branching stems which are cespitose and decum-bent, usually has a tap root,,and leaves that are 2-15 mm wide. Lasthenia macrantha subsp. bakeri (n = 24) has simple; erect stems a r i s i n g from thickened fibrous roots, and narrow leaves 1-2 mm wide. Lasthenia macrantha subsp. prisca (n = 16) has smaller leaves (2). Although a l l three sub- > species share a similar flavonoid chemistry, subsp. bakeri and subsp. prisca have more i n common with subsp. macrantha than they do with one another. Lasthenia macrantha subsp. bakeri i s unique i n the species i n synthesizing four quer-c e t i n glycosides (4). In t h i s s t u d y , , L a s t h e n i a macrantha. subsp. macrantha and subsp. bakeri were the only two taxa to share an i d e n t i c a l protein banding p r o f i l e . Due tot.their three homologous globulin bands, they clustered at c o r r e l a t i o n c o e f f i c i e n t 1.0 i n the globulin dendrogram. By sharing f i v e albumin bands, these two taxa were also most s i m i l a r (albumin c o r r e l a t i o n c o e f f i c i e n t 0.455.)>i.. However, out of a t o t a l . of 28 d i f f e r e n t albumin and globulin bands occurringi.ini.the species Lasthenia macrantha, subspecies prisca possessed only one band (globulin band 6) i n common with subsp. ma-crantha and subsp. bakeri (Table I I I ) . Lasthenia macrantha subsp. prisca was removed from the above two taxa i n a l l three 18 dendrograms, and was most c l o s e l y related to Lasthenia kunthii. In contrast, other studies have shown that species, which have been subdivided on morphological, c y t o l o g i c a l , or other grounds, contain seed proteins which exhibit uniform band patterns within the species. For example, i n the genus Suaeda (Chenopodiaceae) , three v a r i e t i e s of Suaeda maritimd,, d i f f e r e n t i a t e d by morphological and e c o l o g i c a l c r i t e r i a , showed aalmost i d e n t i c a l (92.2% s i m i l a r i t y ) seed protein p r o f i l e s (17). The c l a s s i f i c a t i o n of Lasthenia (Table I) has been described as taxonomically conservative, stressing the phenetic unity of the genus and minimizing the differences which e x i s t within i t (1). The s t r i k i n g s i n g u l a r i t y of the seed protein p r o f i l e exhibited by Lasthenia macrantha subsp. prisca, when compared with those exhibited by the other two subspecies, bakeri and macrantha, suggest that more consideration may need to be given to the biochemical d i v e r s i t y existing within the genus. Lasthenia leptalea (section B u r r i e l i a ) i s c l o s e l y related to members of section Baeria (1) . The fcl?aWn"dfd chemistry of Lasthenia leptalea i s i d e n t i c a l to that of the three subspecies of Lasthenia macrantha i n section Baeria (4). The globulin seed proteins of Lasthenia leptalea are closest to those of Lasthenia macrantha subsp. macrantha and subsp. bakeri at a c o r r e l a t i o n c o e f f i c i e n t of 0.25. However.? Lasthenia leptalea shows the greatest s i m i l a r i t y i n albumin proteins to Lasthenia platycarpha and. Lasthenia kunthii, 19 and clusters with Lasthenia macrantha at 0.154. Taking both types of protein into consideration, Lasthenia leptalea f i r s t clustered with Lasthenia macrantha subsp. macrantha and subsp. bakeri at a c o r r e l a t i o n c o e f f i c i e n t of 0.244, supporting the suggestion that t h i s species i s related to those i n section Baeria. A close r e l a t i o n s h i p between sections Lasthenia and Hologymne i s suggested by t h e i r ooccasional i n c l u s i o n i n a d i s t i n c t genus, Lasthenia sensu s t r i c t o . Supportive evidence for t h i s i s the occurrence of the Hologymne flavonol pattern i n Lasthenia glaberrima (4) . In the albumin dendrogrami}'. Lasthenia glaberrima i s most cl o s e l y a l l i e d to Lasthenia chrysantha (Hologymne), cl u s t e r i n g at a r e l a t i v e l y high value of 0.333, and i t i s also s i m i l a r to Lasthenia glabratassnbs'p. glabrata (Hologymne). With globulins, no such close r e l a t i o n -ship was found. In the combined dendrogram, Lasthenia glabe-rrima i s next closest to Lasthenia glabrata subsp. glabrata, although i t clusters with i t at a very low value of 0.099. Three c l o s e l y related taxa, Lasthenia fremontii, Lasthenia burkei, and Lasthenia conjugens (section Ptilomeris) have been the focus of several recent studies (5, 6,118). On c y t o l o g i c a l and morphological grounds, one proposed evolu-tionary scheme places Lasthenia burkei intermediate to the other two species i n the evolutionary pathway: (Lasthenia fremontii -* Lasthenia burkei -»• Lasthenia conjugens or Lasthenia conjugens -»• Lasthenia burkei Lasthenia fremontii) . On the other hand, some i n t e r s p e c i f i c i h y b r i d progenies between 20 Lasthenia fremontii and Lasthenia conjugens are morphologically indistinguishable from Lasthenia burkei, suggesting the pos s i -b i l i t y that t h i s species i s of hybrid o r i g i n . Flavonoid studies do not o f f e r evidence i n support of either of these alternative schemes, though they also do not negate either of them (4). Lasthenia fremontii has only 1 globulin;, band, while Lasthenia burkei and Lasthenia conjugens have two bands each. Lasthenia fremontii has only f i v e albumin bands while Lasthenia burkei Has eight albumin .bands and Lasthenia con-jugens- has nine. This progression of increasing band comple-x i t y i n both protein fractions may support the f i r s t evolutio-nary scheme, Lasthenia fremontii Lasthenia burkei -* -Lasthenia conjugens. Lasthenia burkei and Lasthenia conjugens c l u s t e r together at a relatively high l e v e l (0.5) i n the globulin dendrogram and are the second most similar taxon pair i n the albumin dendrogram (correlation coefficient0044l7). This close s i m i l a r i t y i s confirmed i n the combined dendrogram. , In a l l three dendrograms, Lasthenia fremontii has l i t t l e i n common witht.the other two members of t h i s c l o s e l y knit phylad. Protein banding patterns of i n t e r s p e c i f i c hybrids may further elucidate r e l a t i o n s h i p s . i n t h i s group. Disc electrophoresis of crude seed-protein extracts has been extremely useful i n elucidating evolutionary r e l a -tionships 'in many genera. In Gossypium, comparison of banding patterns sh.as provided, additional information regarding the genetic o r i g i n of natural a l l o t e t r a p l o i d s (19). Similar Studies c l e a r l y support the amphidiploid o r i g i n of Triticum 21 aestivum from the hybridization of the t e t r a p l o i d Tritiaurn dicoccum and the wild d i p l o i d species Aegilops squarrosa (20) . Seed protein p r o f i l e s f u l l y corroborate morphological, cyto-l o g i c a l , and flavonoid data i n delineating the i n t e r n a l re-lationships of a phylogenetic reticulum i n Phlox containing twelve taxa (21.) . The seed proteins of seven Cfffiea taxa showed a c o r r e l a t i o n with genetic and taxonomic information (22). In the present study, disc electrophoresis of albumin and globulin seed fractions has provided some additional i n f o -rmation about the genus Lasthenia, although the number of clear relationships that arose are few. Dendrograms based on unweighted pair-group clustering of s i m i l a r i t y c o e f f i c i e n t s do not r e f l e c t the sectional taxonomy of the genus. Although the present study would suggest that nearly every species of Lasthenia has a unique array of seed proteins, further.sampling of additional populations may reveal the existence of i n t r a -s p e c i f i c v a r i a t i o n . At present, however, most of the i n t e r -s p e c i f i c a f f i n i t i e s that would be deduced on the basis of seed protein s i m i l a r i t i e s are not i n concordance with the a f f i n i t i e s that have been suggested on the basis of evidence from morphological, c y t o l o g i c a l , and other biochemical studies. BIBLIOGRAPHY 1 Ornduff, R. 1966. A biosystematic survey of the g o l d f i e l d genus Lasthenia (Compositae: Helenieae). Univ. Calif. Publ. Bot. 40: 1-92. 2 Ornduff, R. 1971. A new t e t r a p l o i d subspecies of Lasthenia (Compositae) from Oregon. Madrono 21: 96-98. 3 Saleh, N. A. M., B. A. Bohm, and R. Ornduff. 1971. Flavonoids of Lasthenia conjugens and Lasthenia fremontii. Photochemistry 10: 611-614. 4 Bohm, B. A., N. A. M. Saleh, and R. Ornduff. 1974. The flavonoids of Lasthenia (Compositae). Amer. J. Bot. 61: 551-561. 5 Ornduff, R., B. A. Bohm, and N. A. M. Saleh. 1973. Flavonoids of a r t i f i c i a l i n t e r s p e c i f i c hybrids i n Lasthenia. Biochem. Sys-t. 1: 147-151. 6 Ornduff, R., B. A. Bohm, and N. A. M. Saleh. 1974. Flavonoid races i n Lasthenia (Compositae). Brittonia i n press. 7 Zuckerkandl, E.,and L. Pauling. 1965. Molecules as documents of evolutionary h i s t o r y . J. Theor. Biol. 8: 357-366. 8 Boulter, D., D. A. Thurman, and B. L. Turner. 1966. The use of disc electrophoresis of plant proteins i n systematics. Taxon 15: 135-143. 9 Hunziker, J . H. 1969. Molecular data i n plant systematics. p. 280-312. In: Systematic Biology, Proceedings of an International Conference. Publication 1692, Nat. Acad. S c i . , Washington, D. C. 10 Boulter, D., D. A. Thurman, and E. Derbyshire. 1967. A disc electrophoretic study of globulin proteins of legume seeds with reference to th e i r systematics. New p'hytoi. 66: 27-36. 11 Osborne, T. B. 1909. The Vegetable Proteins. Longmans, Green, and Co., London. Chapter X. 12 Fox, D. J . , D. A. Thurman, and D. Boulter. 1964. Studies on the proteins of seeds of the Leguminoseae - I. Albumins. Phytochemistry 3: 417-419. 13 Boulter, D., E. Derbyshire, J. A.. Frahm-Leliveld, and R. M. P o l h i l l , 1970. Observations oh the cytology and seed-proteins of various A f r i c a n species of Crotalaria L. (leguminosae). New Phytol. 69:" 117-131. 22 23 14 Waddell, W. J , 1956. A simple u l t r a v i o l e t spectrpphotometric method for the determination of protein. J. Lab. Clin. Med. 48: 311-314. 15 Davis, B. J . 1964. Disc electrophoresis I I . Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. 16 Sokal, R, R., and P. H. A Sneath. 1963. Principles of Numerical Taxonomy. Freeman, San Francisco. 17 Ungar, I. A., and J . Boucaud. 1974. Comparison of seed proteins i n the genus Suaeda CChenopodiaceae) by means of disc gel electrophoresis. Amer. J'. Bot. 61: 325-330. 18 Ornduff, R. 1969. The o r i g i n and relationships of Lasthenia burkei CCompositae). Amer. J. Bot. 56: 1042-1047. 19 Cherry, J . P., F. R. H. Katterman, and J . E. E n d r i z z i . 1970. Comparative studies of seed proteins of species of Gossypium by gel electrophoresis. Evolution 24: 431-447. 20 Johnson, B. L. 1972. Seed protein p r o f i l e s and the o r i g i n of the hexaploid wheats. Amer. J. Bot. 59: 952—960. 21 Leyin, D. A., and B. A. -Schaal. 1970. Reticulate evolution i n piilox as seen through protein electrophoresis. Amer. J. Bot. 57: 977-987. 22 Payne, R. C., R. O l i v e i r a , and D. E. Fairbrothers. 1973. Disc electrophoretic investigation of Coffea arabica and C. canephora: general protein and malate dehydrogenase of mature seeds. Biochem. Syst. 1: 59-61. P A R T I I PARTIAL CHARACTERIZATION AND SEQUENCE STUDIES OF SAMBUCUS FERREDOXIN 24 CHAPTER I INTRODUCTION The ferredoxins are low molecular weight proteins which function i n a variety of metabolic systems as electron c a r r i e r s . They constitute one group of a larger class of electron c a r r i e r s and enzymes, the iron-sulphur proteins. Previously c a l l e d non-haeme iron proteins, the i r o n -sulphur proteins are defined as those i n which iron i s bound v i a sulphur-containing ligands. They occur, widely, i n bacteria, plants, and animals. The function of iron-sulphur proteins i n carbon and sulphur metabolism, nitrogen f i x a t i o n , photosyn-thesis, r e s p i r a t i o n , and hormone synthesis i s considered cen-t r a l (1). Although these proteins were recognized but a decade ago, i n t e r e s t i n them has increased dramatically.during the past several, years. One r e s u l t has been the p r o l i f e r a t i o n of t r i v i a l names for the members of t h i s unique class of proteins. Only recently has a systematic nomenclature been developed (Figure 1). The chemical, physical, and b i o l o g i c a l properties of iron-sulphur proteins have been reviewed (1, 4, 5, 6, 7), and a comprehensive t r e a t i s e of t h i s class of proteins i s now available (3). I t i s not surprising that a substantial portion of t h i s excellent two-volume work i s dedicated to ferredoxins because, of the three groups of non-conjugated iron-sulphur proteins (see Figure 1), the ferredoxins are most widely d i s t -ributed and numerous. Iron proteins I Haemoproteins Iron-sulphur proteins Others (e.g. f e r r i t i n , t r a n s f e r r i n , oxygenases) Ferredoxins (fd.) (e.g. chloroplast fd. , adrenal f d . , c l o s t r i d i a l fd. , Pseudomonas putida fd.) "High-potential" iron-sulphur proteins (occur i n some photosynthetic bacteria) Rubredoxins (occur i n one aerobic and some anaerobic bacteria) Conjugated iron-sulphur proteins Iron Molybdenum-iron Molybdenum-iron flavoproteins proteins flavoproteins (e.g. succinate (e.g. Mo-Fe protein (xanthine dehydrogenase) f r a c t i o n of bxidase, nitrogenase) aldehyde oxidase) F i g . 1 Recommended c l a s s i f i c a t i o n of iron-sulphur proteins. Modified from r e f s . 2,3. 27 Ferredoxins have been found i n a wide range of organisms, from the fermentative, aerobic, and photosynthetic bacteria, through the blue-green algae, protozoa, and green algae to a l l higher plants and animals investigated thus far (8, 9). They are r e l a t i v e l y small proteins, consisting of a single polypeptide chain, with molecular weights between 6,000 and 13,000. They contain two to eight atoms of iron and equal amounts of inorganic, (acid-labile) sulphur. Ferredoxins are the most electronegative proteins known. They transfer one or two electrons at redox potentials (-300 to -500 mV at pH 7) close to that of hydrogen gas. Due to t h e i r high content of a c i d i c amino acids, ferredoxins show, a great a f f i n i t y for diethylaminoethyl (DEAE)-cellulose, and t h i s property i s u t i l i z e d i n t h e i r p u r i f i c a t i o n . Ferredoxins are distinguished from the other groups of iron-sulphur proteins (see Figure 1) by having a c h a r a c t e r i s t i c electron paramagnetic resonance (EPR) signal at #=1.94 for the reduced protein (2, 10). The o r i e n -tat i o n and mechanism of t h e i r iron-sulphur active centres have been the focus of an ever-increasing number of biophysical, spectroscopic, and X-ray s t r u c t u r a l studies (reviewed i n r e f s . 3, 11). More general properties of ferredoxins have also been reviewed recently (3, 4, 5, 8, 12, 13). The metabolic roles of ferredoxin electron c a r r i e r s are as diverse as the organisms i n which they have been found. For example, ferredoxins, containing eight atoms of iron and sulphur per molecule, have been is o l a t e d from twelve species of Clostridium (9). They function i n a host of key oxidation-reduction reactions, some of which involve pyruvate, xanthine, 28 sulphite, butyrate, formate, and n i c o t i n i c acid metabolism; and nitrogen f i x a t i o n (14). Another anaerobic, nonphotosynthetic 2 — bacterium, Desulfovibvio gigas, contains a 4Fe-4S ferredoxin which i s required for the reduction of sulphite by molecular 2 — hydrogen, In the aerobic Pseudomonas putida, a 2Fe-2S f e r -redoxin (previously c a l l e d putidaredoxin) acts as a redox pro-t e i n i n a camphor hydroxylating system. The green photosynthetic 2 — bacterium Chlorobium has an 8Fe-8S ferredoxin which catalyzes the carboxylation of acetyl CoA and succinyl CoA to pyruvate and a-ketoglutarate respectively (9). Bovine and porcine adre-nal ferredoxin (formerly referred to as adrenodoxin) functions as a component of a s t e r i o d 11g-hydroxylating system. A f e r -redoxin also containing twoiiron atoms and two sulphides has a si m i l a r function i n porcine testes (9). In a l g a l and higher plant chloroplasts, the photoreduction of the 2Fe-2S 2 ferredo-xin i s the terminal photochemical event (15). Ferredoxin plays a key r o l e i n photosynthesis. Besides being connected to NADP reduction, the photoreduction of ferredoxin i s coupled to oxy-gen evolution and to noncyclic and c y c l i c photophosphorylation. Also i t appears that reduced chloroplast ferredoxin plays a regulatory function i n carbon assi m i l a t i o n by a c t i v a t i n g fruc-tose diphophatase (15). In view of the m u l t i p l i c i t y of physio-l o g i c a l roles already known for ferredoxins, i t w i l l not be con-sidered unusual i f the continued study of t h i s group unveils s t i l l a greater variety of functions. S t r u c t u r a l l y and b i o s y n t h e t i c a l l y , b a c t e r i a l ferredoxins are the simplest electron c a r r i e r s known (16). They have been studied from about 35 d i f f e r e n t species (8,9). Examination of 29 the amino acid compositions of four c l o s t r i d i a l and micrococcal ferredoxins shows that they contain only 14 d i f f e r e n t amino acids, nine of which are common to a l l four organisms. These nine - glycine, valine, alanine, proline, glutamic acid, serine, cysteine, and isoleucine - are i d e n t i c a l with those amino acids which are re a d i l y synthesized i n laboratory conditions simulating IsS-e^ g supposedly primitive Earth environment. S i g n i f i c a n t l y , the f i r s t s i x amino acids were those found i n the Murchison meteorite which f e l l i n A u s t r a l i a i n 1969 (10). These and other considerations suggest that ferredoxins may have been one of the f i r s t proteins formed during p r e b i o l o g i c a l evolution (16,17). Generally, b a c t e r i a l ferredoxins are composed of 54-56 amino acid^residues and have a molecular weight of about 6,000 (9). The amino acid sequences of ferredoxins from eight anaer-obic fermentative bacteria are known and these sequences are ex-tremely homologous (9, 18). The differences which occur be-tween them can be attributed to deletions, i n s e r t i o n s , and point mutations during the evolution of the bacteria (19). Ferre-doxins have been studied from about 20 a l g a l and plant species (9). They a l l have a molecular weight double that of b a c t e r i a l ferredoxins, about 11,000, and contain 96-100.amino acid r e s i -dues. The amino acid sequences of ferredoxins from f i v e plant and a l g a l species are known and these are s t r i k i n g l y s i m i l a r . More i n t e r e s t i n g l y though, the a l g a l and plant sequences are homologous to the b a c t e r i a l ferredoxin sequences when the amino-terminal regions of these various ferredoxins are aligned. A l l present-day ferredoxins may have evolved from a common arche-type by gradual lengthening of the genome (9i?,18, 20, 21). 30 Two intermediate ferredoxins support t h i s hypothesis. Ferredoxin from the green photosynthetic bacterium Chlorobium consists of 60 amino acid residues and has a molecular weight of 6,900. Chvomatium, a purple photosynthetic bacterium, has a ferredoxin containing 81 residues with a molecular weight of 10,000. The. amino acid sequences of both ferredoxins are known and also show homology when aligned with other ferredoxins (9, 18). Comparison of the s t r u c t u r a l and functional c h a r a c t e r i s t i c s of a l l ferredoxins which have been studied suggests the pattern of evolutionary development shown i n Figure 2. Amino acid sequence data are of great value i n the study of evolution (22, 23). Changes i n specific.amino acids i n the sequences of homologous proteins can be used by the application of techniques such as c a l c u l a t i o n of mean base difference per codon to trace the development of these proteins from "primitive" types (19, 24). Amino acid sequence differences i n cytochrome o3 haemoglobin, and fibrinopeptides have been successfully applied i n constructing phylogenetic trees which agree well with those phylogenies deduced by more c l a s s i c a l studies of organisms (19). Recently, a family tree has been constructed from amino acid sequences of 21 higher plant cytochromes a (25). The phylogeny of higher plants i s also being investigated by the study of the amino ac i d sequences of plastocyanin (26, 27). However, the use of ferredoxin i n studying plant evolution i s considered more advantageous (17). B i o l o g i c a l l y , ferredoxins are found i n the widest range of organisms - fermentative obligate anaerobes, f a c u l t a t i v e anaerobes, and aerobes. Technically, ferredoxins are obtained i n higher y i e l d s from plant material than are 31 (Spinaaea) Higher Plants (Soenedesmus) Green Algae t Animals (_Spivulina maxima) Blue-green Algae) t (Desulfovibvio gigas) Sulphate-reducing Bacteria (Rhodospiri Hum rubrum) Purple (Non-sulphur) Photosynthetic Bacteria (Chromatium) Purple CSulphur) Photosynthetic Bacteria (Phlorobium limioola) Green Photosynthetic Bacteria t (Clostridium pasteurtianum) "Primitive" Heterotrophic Bacteria Protoferredoxin Gene F i g . 2 Evolutionary development pattern of ferredoxin based on c h a r a c t e r i s t i c s of the respective protein from each organism. Examples i n parentheses are present-day species representative of the groups shown. Modified from r e f s . 9, 18, and 17. 32 cytochrome o and plastocyanin, thus f a c i l i t a t i n g the sequence determination. The phylogenetic tree of ferredoxins calculated from the amino acid sequences known to date i s shown i n Figure 3. Many more ferredoxin sequences must be obtained before the data can be used to solve plant systematic problems. I t i s hoped that such information w i l l not only help i n our understanding of the evolution of plant f a m i l i e s , but also aid i n c l a r i f y i n g relationships within f a m i l i e s . Ferredoxins from only four angiosperms have been sequen-ced thus f a r , as indicated i n Figure 3. (The amino acid sequen-ces of three of these ferredoxins are presented i n the Appen-dix. ) However, ferredoxins have been.purified and characterized from these additional angiosperms: Zea andCCyperrus (Graminales) , Amaranthus (Chenopodiales), Gossypium (Malvales), and Petrose-linum (Umbellales) (9) . isio Chemosystematic and phytoserological studies of the Caprifoliaceae (Rubiales) have indicated possible trends among the c o n s t i t u t i v e genera from primitive to advanced status i n the family (28, 29). With respect to flavonoid characters, the r e l a t i o n s h i p of Sambucustfco Loniaera and Viburnum, and to the rest of the family as a whole i s of special i n t e r e s t (28). The future a v a i l a b i l i t y of protein . sequence information from these genera w i l l hopefulfy add much to our understanding of the taxonomic relationships within the Caprifoliaceae. Toward t h i s goal, the technical f e a s i b i l i t y of performing amino acid sequence studies with ferredoxins i s o l a t e d from these plants must f i r s t be investigated. The wide d i s t r i b u t i o n of Sambucus and the ready a v a i l a b i l i t y of p l e n t i f u l . l e a f material from 1*1 C A J o 'tr P J ( D ( D * < a. I- 1 o t-tiuq H CD o 3 3 (D rt H H-(D O Hi •a rt H CO (D • (D 0 Hi Hi CD H H fD Cb O H-cn H O rt O fD H H fD a-6 3 Anaerobic ferredoxins Photosynthetic Green Bacteria Photosynthetic Purple Bacteria • Scenedesmus (Green alga) Colocassia esoulenta (Araceae, Arales) Medioago sativa (Leguminoseae, Rosales) Leuoaena glauca (Leguminoseae, Rosales) Spinacea oleracea (Chenopodiaceae, Chenopodiales) C O 34 t h i s member of the Caprifoliaceae suggest i t as an i n i t i a l candidate. The choice of Sambueus as the f i r s t source of f e r -redoxin for continuing protein sequence studies i n higher plants i s also appropriate because, both cytochrome e (25) and plastocyanin (26) have already been sequenced from t h i s genus. It w i l l eventually prove i n t e r e s t i n g to see what evolution-ary relationships these three protein families y i e l d for a single genus. The research described i n t h i s thesis was undertaken to determine whether ferredoxin could be i s o l a t e d from leaves of Sambueus raeemosa L. (redberry elder), and thus add to the growing amount of evidence showing the ubiquity of t h i s i r o n -sulphur protein. The investigation of various methods for the extraction and p u r i f i c a t i o n of ferredoxin from leaf mater-i a l therefore formed an i n t e g r a l part of the work. The development of an e f f i c i e n t mode of preparation of the protein was considered an important goal. The subsequent determination of homogeneity, b i o l o g i c a l a c t i v i t y , and chemical and physical c h a r a c t e r i s t i c s could indicate the.degree of s i m i l a r i t y between Sambueus ferredoxin and other plant ferredoxins. Also, i t was hoped that an investigation of the p r a c t i c a b i l i t y of various strategies for the determination of the sequence of Sambueus ferredoxin would aid i n the future elucidation of i t s primary structure. CHAPTER I I EXPERIMENTAL PROCEDURE 1. MATERIALS A. Plant Material Leaves of Sambucus racemosa L. ssp. pubens (Michx.) House var. avbovescens Gray were co l l e c t e d from the Univer-s i t y of B r i t i s h Columbia Endowment Lands. Only green leaves with no obvious signs of i n f e c t i o n were used. A voucher specimen has been deposited i n the University of B r i t i s h Columbia Herbarium (Herbarium Sheet Accession Number 144617). B. Chemicals and Solvents Diethylaminoethyl(DEAE)-cellulose and t r i s (hydroxy-methyl)amino methane (Tris). were obtained from N u t r i t i o n a l Biochemicals Corporation, of Cleveland, Ohio. Whatman DE 23 c e l l u l o s e powder was obtained from W. and R. Balston (Modi-f i e d Cellulose) Limited, Maidstone, England. Sephadex G-25 Fine (20-80 u dry p a r t i c l e diameter) and Sephadex G-75 (40-120 p) were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Nicotinamide adenine dinucleotide phosphate (NADP), thermolysin (protease type X) , N,N-dimethyl-p-phenylenediamine Cgrade III p u r i t y ) , and dansyl amino acids were a l l purchased from Sigma Chemical Company, St. Louis, Missouri. N,N,N',N'-tetramethylethylenediamine (TEMED) ,.. N,N'-methylenebisacryl-amide, r i b o f l a v i n , and 2-amino-2-methyl-l,3-propanediol were obtained from Eastman Kodak Company of Rochester, New York. 36 Ammonium persulphate and concentrated hydrochloric acid were "Baker Analyzed" reagent grade and were obtained from J . T. Baker Chemical Company, P h i l l i p s b u r g , New Jersey. Ninhydrin, l-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl-Cl), P'-toluenesulphonic acid. (PTSA) , and dimethylf ormamide ( s i l y -l a t i o n grade) were obtained from Pierce Chemical Company, Rockford, I l l i n o i s . . Sodium dodecyl sulphate (SDS), phenyliso-thiocyanate (PITC) i n 1 ml. ampules, N-ethylmorpholine, and t r i f l u o r o a c e t i c acid (TFA) were a l l "Sequenal" grade, from Pierce Chemical Company. Dr. C O . Parkes donated 3-(2-ami-noethyl)indole (AEI) a f t e r i t was converted to the free base from the monohydrochloride form, which was also purchased from Pierce. Potassium hydrogen phthalate., 1,10-phenanthroline, and ferrous ammonium sulphate were c e r t i f i e d ACS grade, Fisher S c i e n t i f i c Company, F a i r Lawn, New Jersey. Cyanogum 41 and pyridine ( a n a l y t i c a l reagent grade) were obtained from B r i t i s h Drug Houses (Canada) Limited, Toronto, Ontario. Fluorescamine [4-phenylspiro(furan-2[3HJ,1 1-phthalan)-3,3 1dionej was donated by Dr. C. 0. Parkes and was obtained from Hoffmann-La Roche (Canada) Incorporated, Montreal, Quebec. The protein molecu-l a r weight standards used i n SDS electrophoresis were also donated by Dr. Parkes, and were obtained from the same sup-p l i e r s as noted by Eng and Parkes (30). Ethyleneimine was purchased from Koch-Light Laboratories Limited, Colnbrook, England. Trypsin-L- (l-tosylamido-2-phenyl)ethyl chloromethyl ketone (Trypsin-TPCK) was obtained from Worthington Biochem-i c a l Corporation, Freehold, New Jersey. For dansylation, Pyrex culture tubes without rims, 6 x 50 mm, (9820) were 37 purchased .from..Corning Glass Works.,.. Corning., New York. Poly-amide sheets- were obtained from Cheng Chin Trading Company, Tapei, Taiwan. Nitrogen was L grade (20 ppm oxygen) and was obtained, from Canadian Liquid A i r Limited,, Vancouver, B r i t i s h Columbia. A l l other chemicals were reagent grade. 2. METHODS A. Preparation of Column Chromatographic Media (1) DEAE-Cellulose DEAE.-cellulose was precycled and equilibrated before use, according to standard procedures (31). The dry c e l l u l o s e powder (60 g) was s t i r r e d into 1 l i t e r of 0.5 M HC1 and l e f t to stand for 1 hr with occasional s t i r r i n g . The swollen gel was f i l t e r e d and washed with deionized water i n a large Buch-ner funnel using a water aspirator. When the pH pf the f i l -t r ate reached 4.0, the moist bed of c e l l u l o s e was s t i r r e d for 2 hr i n 1 l i t e r of 0.5 M NaOH, f i l t e r e d and washed as before. The 0.5 M NaOH treatment was repeated.. The neutral exchanger was then s t i r r e d into 1.5 l i t e r s of deionized water i n a 4 l i t e r Buchner f l a s k . The pH of the slurry was adjusted to 3.0 with 1 M HC1.- The DEAE-cellulose was degassed by s t i r r i n g i t magnetically under water pump vacuum u n t i l b o i l i n g occur-red. I t was t i t r a t e d to pH 7.5 with saturated T r i s solution. Fine p a r t i c l e s were removed by suspending the s t i r r e d s l u r r y i n a 2 l i t e r graduated cylinder, l e t t i n g i t s e t t l e for 1 hr, 38 and siphoning off the supernatant. This was.repeated 5 times. Each time, the volume of the s l u r r y was made up to 2 l i t e r s with deionized water.. The exchanger was then, equilibrated" by: (al suspending i t i n the. appropriate buffer, (b) t i t r a t -ing i t to pH 7.5 with the .appropriate buffer component, and (c) f i l t e r i n g i t i n a Buchner funnel. Steps a, b, and c were repeated u n t i l the pH of the f i l t r a t e remained for at lea s t 2 successive washes. The DEAE-cellulose was f i n a l l y degassed and stored i n the cold room.(4 C). Columns were poured i n the cold room immediately p r i o r to use, and were s t a b i l i z e d by running e q u i l i b r a t i o n buffer through them. (2) Sephadex Gels Sephadex.gels were boiled for 1 hr i n excess buffer, l e f t to stand overnight, and degassed. Columns were poured, s t a b i l i z e d , equilibrated, stored, and t h e i r void volumes de-termined according to standard methods (3.2) . B. Preparation of Ferredoxin (1) Preparation of Buffers Buffers were made with d i s t i l l e d water and t i t r a t e d to the correct pH. Nitrogen was bubbled through them for 5 min. After degassing (water pump), and adding g-mercapto-ethanol i f necessary, the pH was checked. Buffers were stored 39 i n the cold room at 4 C. Throughout.the extraction procedure a l l solutions- were adjusted.to pH 7.5. (2) I n i t i a l T r i a l s I n i t i a l l y the procedure of Keresztes-Nagy and Margo-l i a s h (33) was chosen for the p u r i f i c a t i o n of ferredoxin from Sambucus.leaves because i t did .not involve the use of organic solvents,.large-scale centrifugation, d i a l y s i s , or p r e c i p i t a -t i o n . But aft e r several t r i a l s , the method proved unsuccess-f u l . After homogenization. and f i l t r a t i o n through cheesecloth, the aqueous extract did not pass through a large bed of DEAE-ce l l u l o s e because a l i p i d - l i k e residue coated the top of the re s i n bed and r e s t r i c t e d the flow. I t seemed possible to ov-ercome t h i s problem by using the procedure of Petering and Palmer (34). The aqueous cheesecloth f i l t r a t e was treated with DEAE-cellulose by batch technique, but the separation and subsequent washing of the r e s i n was laborious, due to the u n a v a i l a b i l i t y of a 6 l i t e r centrifuge. Also, when the wash-ed res i n was poured into a column for e l u t i o n of adsorbed f e r -redoxin, the flow rate was p r o h i b i t i v e l y slow. Since wax-l i k e material from the aqueous leaf extract was the apparent cause of poor chromatographic performance, a new procedure using acetone was employed. The method of Crawford and Jen-sen (35), with some modifications, proved successful. 40 13.) Purification of Sambueus Ferredoxin The entire preparation was carr i e d out i n the cold room C4 C). At anytime at which i t . was necessary to i n t e r -rupt, the ^preparation, the solution containing ferredoxin was flushed with and stored under nitrogen. A l l solutions, ex-cept 50% acetone-water, contained 1 mM g-mercaptoethanol. a. Homogenization Fresh Sambueus leaves were co l l e c t e d , immediately t a -ken into the cold room and the petioles removed. Leaflets (400 g) were placed loosely i n a 5 l i t e r Waring Blendor and homogenized with 800 ml of 50% acetone-water (-20 C) con-taining 1.8 g T r i s . A fine s l u r r y was obtained, usually within 3 min, by varying the speed of the blendor using a voltage regulator. Homogenization was. continued for 2 min at high speed. The homogenate was squeezed through 4-ply cheesecloth i n a large Nalgene funnel and the f i l t r a t e was col l e c t e d i n a 1 l i t e r f l a s k . The pH of the cheesecloth f i l t r a t e was adjusted'to 7.5 with cold 1 M NaOH or 1 M HC1 i f necessary. The f i l t r a t e was poured into s i x 250 ml Nal-gene centrifuge bottles and centrifuged at 20,000 x g for 10 min at -15 C. b. DEAE-Cellulose Adsorption The dark green supernatant was decanted into a 1 l i -ter f l a s k . I t was then siphoned through a 6 x 2 cm bed of DEAE-cellulose, equilibrated with 2 mM Tris-HCl, i n 50% acetone-41 water, pH 7.5, in.a 200 ml coarse sintered disc funnel. The flow rate was about 2 0 ml/min. The bed was washed with 10 mM Tris-HCl, pH.7.5 u n t i l the e f f l u e n t was clear (ca. 2 l i t e r s ) . The top of the bed was exposed and buffer 0.1 M Tris-HCl, 0.8 M NaCl, pH 7.5) was c a r e f u l l y pipetted on. The adsorbed protein was slowly eluted i n about 100 ml of red-brown s o l -ution. c. DEAE-Cellulose Chromatography The ferredoxin-containing solutions, from two 400 g l o t s of leaves, were combined i n a Mariotte b o t t l e , and d i -luted with 4 parts of 1 mM 3-mercaptoethanol. The solution was readsorbed on a 2.5 x 45 cm DEAE-cellulose column, equi-l i b r a t e d with 0.1 M Tris-HCl, 0.2 M NaCl, pH 7.5 buffer. The column was washed with.1.5 l i t e r s of t h i s buffer. The flow rate was 4 ml/min. A d i f f u s e yellow red. band containing the ferredoxin separated from the dark brown.zone retained at the top of the column. The column was further developed with 500 ml of 0.1 M Tris-HCl, 0.3 M NaCl, pH 7.5., Twenty ml f r a c -tions were co l l e c t e d and scanned from 210 to 450 nm using a Unicam SP1800 recording spectrophotometer. Ferredoxin eluted as a d i s t i n c t red-brown solution, showing a peak at 330 nm, usually contained i n the 1630-1720 ml f r a c t i o n . The e f f l u e n t p r i o r to and following the ferredoxin band was l i g h t yellow. d. Sephadex G-75. Chromatography The fractions.containing ferredoxin were pooled and d i l u t e d 1:3 with 1 mM 3-mercaptoethanol. The solution was concentrated by readsorption on a 3 x 1 cm bed of Whatman DE 23 c e l l u l o s e , equilibrated with 10 mM Tris-HCl, pH 7.5, i n a 45 ml coarse sintered disc funnel and by slow e l u t i o n with 0.1 M Tris-HCl, 0.8 M NaCl, pH 7.5 buffer. The concentrated ferredoxin solution (ca. 20 ml) was siphoned through a 3-way valve on to a 4.7 x 100 cm Sephadex G-75 column and eluted with 10 mM Tris-HCl, 0.2 M NaCl, pH 7.5. The flow rate was 60 ml/hr with a hydrostatic pressure of. 30 cm. The sharp red band of pure ferredoxin separated from two slower moving l i g h t green bands, and had an el u t i o n volume CVg) of 1160 ml. e. Desalting and Lyop h i l i z a t i o n The pure ferredoxin solution was concentrated as i n step (dl above, except there was no need for p r i o r d i l u t i o n . The concentrated ferredoxin solution (ca. 20 ml) was put on a 4 x 20 cm column of Sephadex G-25 Fine and eluted with d i s t -i l l e d water. Fractions absorbing at 280 nm were pooled and freeze-dried. The brown-red l y o p h i l i z e d protein was stored desiccated at -20 C. C. .Determination o f t h e Molar Extinction C o e f f i c i e n t of Sambueus Ferredoxin Sambueus ferredoxin was p u r i f i e d as rapidly as pos-s i b l e from fresh leaves. The concentrated protein from p u r i f i c a t i o n step (e) (see t h i s chapter, section B3), was 43 dissolved quantitatively i n 25 ml of 0.1 M Tris-HCl, 0.8 M NaCl, 1 mM 3-mercaptoethanol, pH 7.5, and kept on i c e . The o p t i c a l densities at 420 nm, of two 3 ml samples, were read i n a Unicam SP1800 redording spectrophotometer and the re s u l t s averaged. The ferredoxin solution was then desalted and freeze-dried. The dry weight was used to calculate the con-centration of the solution. The extinction c o e f f i c i e n t could then be calculated d i r e c t l y . D. Assay of Sambucus Ferredoxin A c t i v i t y The procedure of Crawford, and Jensen (35) u t i l i z i n g spinach chloroplasts was modified to determine the a c t i v i t y of Sambucus ferredoxin. Chloroplasts were prepared by homo-genizing fresh spinach leaves (50 g) with 70 ml of 0.25 M Tris-HCl, 0.5 M s o r b i t o l , 5 mM 8-mercaptoethanol, pH 7.5, for 30 sec i n a Waring Blendor and f i l t e r i n g the homogenate through 2-ply cheesecloth and glass wool. The chloroplasts were co l l e c t e d according to Keresztes-Nagy and Margoliash (33); chlorophyll content was determined according to Arnon (36). The sample of Sambucus ferredoxin, in.10 mM Tris-HCl, 0.2 M NaCl, 1 mM 3-mercaptoethanol, pH 7.5, was taken from the Sephadex G-75 column e f f l u e n t (see t h i s chapter, section B3, p u r i f i c a t i o n step d) which showed maximum absorbance at 280 nm. The amount of protein used i n the reaction mixture was calculated using, the extinction c o e f f i c i e n t for Sambucus ferredoxin (see t h i s chapter, section C). The rate of l i g h t -44 dependent reduction of NADP was measured i n a cuvette contain-ing spinach, chloroplast suspension equivalent to 0.1 mg of chlorophyll, 0.1 mg of Sambucus ferredoxin, and 2 uM of NADP i n 3.0 ml of 0.25 M Tris-HCl, 0.5 M s o r b i t o l , 5 mM 3-mercapto-ethanol, pH 7.5 buffer. The reaction mixture and the blank, i n which the ferredoxin sample was replaced with -the above 10 mM Tris-HCl buffer, were illuminated at 20 C l i g h t from a 100 watt incandescent bulb, and were protected from excess heat by a 5-cm thick water f i l t e r . The absorbance of the reaction mixture at 340 nm was read against the blank af t e r successive 3-min periods of ill u m i n a t i o n . Calculation, of unit and s p e c i f i c a c t i v i t y followed the d e f i n i t i o n s of San Pietro C37). One unit of enzyme i s that amount producing a change of 1.0 O. D. i n 10 min at 340 nm, when the reaction mixture contains 0.1 mg chlorophyll per 3.0 ml. This unit represents the reduction of 4.8 uM of NADP per mg of chloro-p h y l l i n 10 min. E. Polyaerylamlde Disc Electrophoresis The purity of Sambucus ferredoxin, prepared according to the procedure outlined i n section B o f . t h i s chapter, was assessed by disc electrophoresis i n 7.7% polyacrylamide gels, pH 9.0. The general procedures and materials used were those described by Ornstein and Davis i n th e i r 1962 preprint(38). After small-pore and spacer gel polymerization, samples con-taining about 60 jag of ferredoxin in. 0.1 ml of 0.1 M Tris-^ 45 HC1, 0.8 M NaCl, 1 mM 3-mercaptoethanol, pH 7.5, were layered on top of the spacer gel i n the absence of any further a n t i -convection agents, such as 10% sucrose.. Samples were run at 4 C at 4 ma per tube for 10 min and then at 6 ma per tube for a further 65 min. After staining for 2 hr i n a 1% solution of Buffalo Blue Black i n 7% acetic acid, gels were destained and stored i n 7% acetic acid. F. SDS Electrophoresis Estimation of molecular weight'followed the procedure of Eng and Parkes (30). Ten microgram samples of l y o p h i l i z e d Sambueus ferredoxin were dissolved i n 100 u l i t e r s of diluent buffer and complexed with SDS f o r 5 min at 100 C. Fluoresca-mine (5 u l i t e r s , 1 mg/ml acetone) was added and the mixture shaken. Protein standards (ovalbumin, chymotrypsinogen, myo-globin, lysozyme, and ribonuclease) were prepared the same way. Polyacrylamide gels, prepared using Cyanogum 41 and prerun for 30 min at 4 ma/gel, were used for electrophoresis of labeled proteins. G. Amino Acjd Analysis (.1) Automatic Amino Acid Analysis The amino acid composition of Sambueus ferredoxin was determined on the Beckman Spinco Model 120C automatic amino 46 acid analyzer according to the method of Spackman, Stein, and Moore (39). The analyzer, equipped with an expanded range card, had a detection l i m i t of 4.0-4.5 mv. Approximately 20 nmoles of each amino acid i n the protein yielded peaks of adequate s i z e . Freeze-dried ferredoxin was dissolved i n d i s t i l l e d water to make a 0.4% solution. One hundred micro-l i t e r samples of t h i s solution were hydrolyzed with 100 u l i t r e s of concentrated HC1 (Baker Analyzed) i n evacuated, sealed tubes at 104-108 C. Duplicate samples were hydrolyzed for 19, 38, and 62 hours to enable extrapolation to max-imum yi e l d s f o r degradable residues and to enable an equally v a l i d assessment of slowly hydrolyzed residues such as valine and isoleucine. After hydrolysis, one set of tubes was cool-ed, opened, and dried over NaOH p e l l e t s and P 2 O 5 i n a desic-cator with o i l pump vacuum.. The dried hydrolyzates were d i s -solved i n 2.0 ml of 0.2 N sodium c i t r a t e buffer, pH 2.2, according to standard procedure. The duplicate set of tubes was cooled, opened, and made up to 2.0 ml with the above sodium c i t r a t e buffer, according to Robel, to check for losses of c e r t a i n amino acids due to adsorption to the glass tubes during desiccation (40). In each case, 200 u l i t r e s of the sample was placed on the short column (5.7 cm) to resolve basic residues, and 200 u l i t r e s was placed on the long col-, umn (57.7 cm) to resolve the neutral and a c i d i c residues. Two hundred m i c r o l i t r e s of i n t e r n a l standard solution (a-amino-|3-guanido-propionic acid for the basic amino acids and norleucine for the a c i d i c and neutral amino acids) was 47 included with each run to enable determination of ninhydrin deterioration. Analysis of S-3-aminoethylcysteinylferredoxin (AECys-ferredoxin) (see t h i s chapter, section 1(2)) followed the above procedures, except that the short column r e s i n height was 17 cm instead of 5.7 cm. S-3-aminoethylcysteine was eluted between the positions of lys i n e and h i s t i d i n e . The integration constant for AECys was taken to be 91.6% of that for l y s i n e , as suggested by Hofmann (41). (2) Determination of Tryptophan The determination of the tryptophan content of Sam-bueus ferredoxin followed the method of Liu.and Chang (42), as described by L i u (43). A solution of 3.0 N p-toluenesul-phonic acid (PTSA) containing 0.2% 3-(2-aminoethyl)indole (AEI) was prepared by adding deionized water to the reagents i n a 1 ml volumetric test tube. The mixture was heated i n a b o i l i n g water bath u n t i l a l l of the AEI dissolved, where-upon the solution became pink. Freeze-dried ferredoxin CO.3 mg) was hydrolyzed with 100 u l i t r e s of the above s o l -ution i n an evacuated, sealed tube for 24 hours at 110 C. After hydrolysis, 0.4 ml of 0.5 M NaOH was added and the con-tents of the tube mixed. Aliquots (250 u l i t r e s ) were ana-lyzed on the Beckman Spinco Model 120C automatic amino acid analyzer (see t h i s chapter, section Gl) using a 17 cm re s i n column. Using 0.35 N sodium citr a t e , buffer, pH 5.25, tryp-tophan was el'ufced. before lysine,, with, an elu t i o n volume of 48 81 ml. Calculations were based on the analysis of a 20 nM standard solution of tryptophan. H. Determination of Non-Haeme Iron The non-haeme iron content of Sambueus ferredoxin was determined by the c-phenanthroline method of,Harvey, Smart, and Amis (44) as described by Matsubara (45). Samples, 0.2 ml of 0.1 M Tris-HCl, 0.8 M NaCl, 1 mM 3-mercaptoethanol, pH 7.5, containing 0.01-0.02 umole of ferredoxin, were heat-ed with 1.3 ml of 1% HC1 for 5 min at 80 C i n 12 ml conical centrifuge tubes with glass marbles on top to reduce evapo-ra t i o n . The tubes were then centrifuged for 10 min at 800 x g and 1 ml of each supernatant was pipetted into 13 x 100 'mm test tubes. These aliquots were mixed with 0.5 ml of 0.2 M potassium hydrogen phthalate.in 0.3 M NaOH, 1 ml of 0.5% 1,10-phenanthroline i n water,, and 1 u l i t r e of 8-mercaptoetha-nol. The absorbance at 511 nm was measured.. Ferrous ammonium sulphate, dissolved in. the above Tris-HCl buffer, was used to obtain a standard curve. Carried through t h i s procedure, 0.1 yg atom of ir o n gave an absorbance of 0.42 i n 2.5 ml reaction volume. I. Protein Modification (1) T r i c h l o r o a c e t i c Acid Treatment The procedure of Matsubara, Sasaki, and Chain was f o l -49 lowed to remove iron and l a b i l e sulphur from native Sambucus ferredoxin (46). Freeze-dried . p r o t e i n (50 mg) was dissolved i n 5 ml of 0.05 M NH40H pH 10.3, i n a 14 ml So r v a l l c e n t r i -fuge tube, and treated with 5 ml of cold 1.2 M tr i c h l o r o a c e -t i c acid (TCA). After standing i n the r e f r i g e r a t o r for 1 hr, the p r e c i p i t a t e was col l e c t e d by centrifugation at 27,000 x g for 10 min at 4 C. The supernatant was removed c a r e f u l l y . The pale yellow brown p r e c i p i t a t e , resuspended using a thi n glass s t i r r i n g rod, was washed with 10 ml of cold 0.6 M TCA and then redissolved i n 5 ml of 0.1 M NaOH. Treatment with 1.2 M TCA was repeated, and the pr e c i p i t a t e was successively washed with 10 ml each of 0.6 M TCA, acetone, and anhydrous ethyl ether. The ether, supernatant was c a r e f u l l y removed and the protein was dried i n a desiccator. The pr e c i p i t a t e was pulverized with the s t i r r i n g rod into a f i n e , dry powder during drying. (2) Reduction and S--3-Aminoethylation To convert native Sambucus ferredoxin to the S-3-amino-eth y l c y s t e i n y l (AECys) derivative, the method of Raftery and Cole (47), modified by Matsubara et. at. (46), was used as described below. T r i c h l o r o a c e t i c acid-treated ferredoxin (44 mg) was dissolved i n 3.3 ml of 7 M urea ( t i t r a t e d to pH 8.05 with 0.01 M NaOH) i n a 50 ml Nalgene centrifuge tube. Octanol (5 u l i t r e s ) was added and nitrogen was bubbled through the solution for 5 min, at which time 0.55 ml of 3-mercapto-ethanol was- added. The tube was flushed with nitrogen for a o 50 further 5 min, sealed with Parafilm, and.the solution was incubated at 40 C for 4.5 hr. The pH was brought to 9.8 by-adding 1.144 g of 2-amino-2-methyl-l,3-propanediol. A th i n combination electrode connected to a Radiometer pH meter, was inserted into the solution. The. reduced ferredoxin was t r e a t -ed with 0.8 ml of ethyleneimine i n 0.2 ml aliquots over a period of 10 min, during which time the solution was being flushed with nitrogen, s t i r r e d with a small magnetic bar, and t i t r a t e d with concentrated HC1 to maintain pH 9.0 (ca. 0.55 ml added i n t o t a l ) . The reaction was allowed to proceed f o r a further 20 min at 40 C i n a nitrogen atmosphere. Eleven ml of 0.8 M t r i c h l o r o a c e t i c acid, and enough 1.6 M TCA (ca. 2.5 ml) were added i n order to p r e c i p i t a t e the derivative. After 30 min at 4 C, the p r e c i p i t a t e was c o l l e c t e d by c e n t r i -fugation at 27,000 x g for 10 min at 4 C. The p e l l e t was resuspended i n and washed with 25 ml of 0.6 M TCA. I t was resuspended i n deionized water and brought to pH 10.1 with 6 M NHljOH, i n a t o t a l volume of 35 ml. The dissolved protein was dialyzed against 2 x 4 l i t r e s of deionized water for 18 hr and l y o p h i l i z e d . J . Digestive Procedures with Thermolysin and Trypsin C D Digestion of TCA-Treated Ferredoxin with Thermolysin T i t a n i et. al. reported that commercial preparations of thermolysin were not homogeneous by either disc gel elec-trophoresis or NH2-terminal analysis (48). To minimize pos-s i b l e contamination of ferredoxin peptides on high voltage electrophoresis papers, thermolysin was p u r i f i e d , under con-diti o n s which prevented autolysis, before i t was used i n d i -gestive procedures. However, t r i a l digests with enzyme pur-i f i e d by gel f i l t r a t i o n through Sephadex G-75 i n 0.2 M am-monium acetate pH 6.0, and subsequent separation of peptides, showed no marked improvement i n r e s u l t s . Indeed, an apparent decrease i n the rate of digestion of ferredoxin was observed with t h i s thermolysin preparation, when compared with the or-i g i n a l commercial preparation. Thermolysin (Sigma protease type X, l o t 23C-0280) was therefore used d i r e c t l y , without further p u r i f i c a t i o n . The enzyme:substrate r a t i o (1:120, w/w) of Rao and Matsubara was used (49). A t y p i c a l digestion was done i n 3.0 ml of 0.2 M ammonium acetate, pH 8.05, containing 18 mg of TCA-treated Sambucus ferredoxin and 0.15 mg of thermo-l y s i n . The sample was incubated at 40 C for 4 hr i n a 14 ml So r y a l l centrifuge tube. At 1 hr i n t e r v a l s , 4 u l i t r e aliquots were spotted on pH indicator paper, and the reaction mix-ture was adjusted to pH 8.0 with 2 M NH^OH i f necessary. Digestion was stopped by adding one drop of acetic acid. The digest was chromatographed d i r e c t l y on Sephadex G-25 Fine. (2) Tryptic Digestion of AECys-Ferredoxin Matsubara and Sasaki reported a t r y p t i c digestion of spinach AECys-ferredoxin, i n which the substrate (180 mg) was 52 dissolved i n 20 ml of water, adjusted to pH 8.2 with 0.1 M NaOH, and digested with 5 mg of enzyme for 4.5 hr. The pH was maintained at 8.2 during the reaction (50). In contrast, Sambueus AECys-ferredoxin proved insoluble at pH 8.2. Accor-dingly, digestion was carr i e d out with the following modifi-cations. Deionized water (2 ml), brought to pH 8.0 with 0.1 M NaOH, was added to 27 mg of AECys-ferredoxin, i n a 13 x 100 mm tes t tube. Afte r r a i s i n g the pH to 10.0 with 1 M NaOH, most of the protein dissolved, and the mixture was s t i r r e d magnetically for 2 hr. The pH was lowered slowly to 8.9 with 0.1 M HC1, and considerable p r e c i p i t a t i o n occurred. TPCK-trypsin (0.27 mg) was added to the s t i r r e d solution and the digest was incubated at 37 C. After 3.5 hr, the solution had cleared s l i g h t l y , and the pH had f a l l e n to 8.2. The s o l -ution was adjusted to pH 8.9 with 0.1 M NaOH, and another 0.27 mg quantity of TPCK-trypsin was added. Digestion was f i n a l l y stopped.after a further 2.5 hr by lowering the pH to 5 with acetic acid. A voluminous p r e c i p i t a t e appeared aft e r 30 min at 20 C. The sample was centrifuged at high speed i n a c l i n i c a l centrifuge. Both p e l l e t and supernatant were re-tained 21 the l a t t e r being applied d i r e c t l y to Sephadex G-25 Fine for gel f i l t r a t i o n . K. Column Chromatographygof Enzyme Digests Thermolytic and t r y p t i c digests were fractionated by the following procedure. Samples CI ml charge volume) were made 5% Cw/v) i n sucrose; layered on a 1.4 x 97 cm column of 53 Sephadex G-25 Fine, and eluted with 10 mM NH^OH, pH 10. The flow rate was 8 ml/hr with a hydrostatic pressure of 29 cm. Fractions (2.5 ml) were c o l l e c t e d using an ISCO Model 270 f r a c t i o n c o l l e c t o r , and t h e i r absorbance at 220 and 280 nm was measured. A l l of the digest was fractionated by repeating chromatographic runs i n t h i s way. Appropriate fractions were pooled and concentrated by rotary evaporation i n 50 ml flasks at 30 C. Concentrated samples (ca. 3 ml) were then ready for preparative peptide p u r i f i c a t i o n by high voltage paper e l e c t r o -phoresis. L. High Voltage Paper Electrophoresis of Peptides High voltage electrophoresis of peptides was used as the f i n a l p u r i f i c a t i o n step i n the protein digest i s o l a t i o n procedures. The v e r t i c a l striphhorgh voltage electrophoresis apparatus used was custom-built and of the Michl type. The operation of t h i s system was s i m i l a r to that described by Ryle et. al. (51). Electrophoresis at both pH 6.5 and pH 1.9 was performed i n glass Chromotanks u t i l i z i n g xylene and varsol respectively as i n e r t coolants. Buffers used were as follows: Constituents Ratios by volume pH 6.5 Pyridine-acetic acid-water (100:4:900) pH 1.9 Formic acid-acetic acid-water (9:12:179) Whatman 3MM f i l t e r paper was used for separation of peptides. The sample loads (100 nmoles/cm) suggested by Ambler (52) were considered to be maximum. For the f i r s t dimension, at pH 6.5, electrophoresis was carr i e d out on f u l l length sheets of 3MM for 4 0 min at 3 KV. The sheets were dried and s t r i p s cut from them were sewn t o new sheets of 3MM. The second dimen-sion, at pH 1.9, was run perpendicularly to the f i r s t f or 35 min at 3 KV. The in t e r n a l fluorescent markers of Brown and Hartley were included with each run to act as guides, for location of peptide spots (53). The papers were dried and the fluorescent bands outlined in. p e n c i l . Peptides were i n -i t i a l l y located by dipping papers i n cadmium-ninhydrin reag-ent (54) f and heating them i n a warm oven for about 5 min, u n t i l the i n i t i a l colours of the peptide spots developed. The papers were then stored i n in d i v i d u a l p l a s t i c bags i n the dark at 20 C overnight; (55). Changes i n spot colour gave some in d i c a t i o n of NH2-terminal residues. Peptide spots on unstained papers were located by c i r c l i n g areas of correspon-ding m o b i l i t i e s . These spots were cut out and stitched to short s t r i p s of 3MM paper. P u r i f i e d peptides were then eluted into 10 x 75 mm t e s t tubes using the eluting solvents of Shotton and Hartley (55). Peptides were evaporated to dryness covered with Parafilm, and stored i n the freezer. M. NH 2-Terminal and Sequence Determination The general procedure of Gray (56) asmmd.di/'fied by Hart ley C 5 7 ) was used for NH 2-terminal determination and sequence of peptides and protein. This "Dansyl-Edman" method allowed f 55 minimum loss of material for NH 2-terminal determination-and maximum speed of degradation. (1.) Dansylation of Peptides The method of Gray was followed (58) f although some of the volumes of reactants were modified. Dansyl tubes were cleaned before use by heating them at 500 C i n a muffle f u r -nace overnight and then cooling. After each addition, the dansyl tubes were centrifuged i n a bench-top centrifuge at high speed for 30 sec to ensure that a l l of the reactant was at the bottom of the tubes. The peptide, i n the 10 x 75 mm tube (see thi s chapter, section L), was taken up i n 200 u l i t r e s of aqueous pyridine (50% v/v, flushed with N2) and an appro-pri a t e sample (5-50 u l i t r e s ) , depending on the concentration, was removed and transferred to a dansyl tube. The sample was evacuated to dryness i n a heated aluminum block which was f i t -ted with a desiccator cover, according to Laycock (59). Five m i c r o l i t r e s of 0.2 M NaHC03 were added and the sample was dried down again. Deionized water (5 u l i t r e s ) and 5 u l i t r e s of dansyl chloride solution (2.5 mg/ml i n acetone) were added to the sample which was then covered with Parafilm and incu-bated at 45 C for 20 min. The sample was evaporated and then hydrolyzed with 20 u l i t r e s of 6 N HC1. The. tube was evacuated and sealed to prevent any possible oxidative degradation. Hydrolysis at 105 C was performed routinely for 6 hr, although v a l i n e - and isoleucine-containing peptides often required 20 hr. Tubes were opened and the contents were dried under vacuum. 56 (2) Dansylation of Protein The procedure recommended for proteins by Gray (58) was followed with s l i g h t modifications. Native ferredoxin was dissolved i n 50% aqueous pyridine, whereas AECys-ferredoxin was suspended insNeethy/lmorphoilne^pyridine^waterd?.,- ". . ' (2:9:9, by volume). Approximately 50-100 ug of protein was transferred to a dansyl tube and dried as above. After add-ing 50 u l i t r e s of 1% (w/v) SDS, the sample was heated i n a b o i l i n g water bath for 5-7 min. N^Ethylmorpholine (50 u l i t r e s ) was added to the cooled solution, and mixed thoroughly with a Vortex mixer. A dansyl chloride solution (2.5 mg/100 u l i t r e s of dimethylformamide) was prepared and 75 u l i t r e s of t h i s was added with thorough mixing. The dansyl tube was covered with Parafilm and l e f t at 20 C overnight. Acetone (0.5 ml) was added and the labeled protein p r e c i p i t a t e d at 4 C for 1 hr. The material was centrifuged and the supernatant was c a r e f u l l y poured o f f . The p r e c i p i t a t e was washed with 0.5 ml of 90% acetone, centrifuged, and dried. Hydrolysis was c a r r i e d out ass described for peptides i n section M;(.l)ibabove. (3) I d e n t i f i c a t i o n of Dansyl Amino Acids Thin-layer chromatography of dansyl amino acids on polyamide sheets was performed e s s e n t i a l l y according to the method of Hartley (57) using some modifications of Olafson (60) and Weiner, P i a t t , and Weber (61). Each dried dansyl hydrolyzate was taken up i n 2.5 u l i t r e s of 50% aqueous pyr-idi n e . Using a, 1 u l i t r e Drummond c a p i l l a r y pipet, 0.5 p l i t r e s 57 of sample were applied to both sides of a 5 x 5 cm poly-amide sheet, 8 mm i n from the edge. The o r i g i n spot was not allowed to exceed 2.5 mm i n diameter. Standard marker solu-tion (0.2 u l i t r e s ) was applied to one side. Plates were dev-eloped i n 250 ml beakers (two plates per beaker) with about 5 ml of solvent i n each. Ascending chromatography i n two dimensions was performed using the standard solvent systems. System 1: 1.5% formic acid (v/v) System 2: Benzene-acetic acid (9:1, v/v) System 3: Ethyl acetate-methanol-acetic acid (20:1:1) by volume System 4: 0.1 M ammonia-ethanol (9:1, v/v) System 4 was sometimes used to resolve a-dansyl h i s t i d i n e , dansyl arginine, and dansyl lysine (62) . Systems (3) and (4) were run i n the same d i r e c t i o n as system (2), which was per-pendicular to the d i r e c t i o n of system (1). Unknown dansyl amino acids were i d e n t i f i e d i n u l t r a - v i o l e t l i g h t by the re-latio n s h i p of t h e i r m o b i l i t i e s to those of the standards. i (.4) Edman Degradation a. Coupling with Phenylisothiocyanate (PITC) The modified Edman degradation procedure described by Gray (63) was used. The volume of the peptide solution was restored to 200 y l i t r e s (see section M(l) above) with 50% aqueous pyridine. If protein was being degraded, the sample (in a 13 x 100 mm test tube) was made up to 500 u l i t r e s with the appropriate solvent (see section M(2) above). A h a l f -58 volume of 10% PITC i n pyridine (prepared fresh every two weeks, stored under N 2 i n the freezer) was added, and the tubes were flushed with N 2 , covered with Parafilm, and incubated at 50 C for 30 min. The reaction was stopped by evaporating the sam-ples to dryness i n a 60 C desiccator with NaOH p e l l e t s and P 20 5. The desiccator was evacuated using a dry ice-acetone trap and an o i l pump. When the tubes were dry, the P 20 5 was replaced, and the evacuated desiccator was kept at 60 C for a further 10 min. Water, pyridine, unreacted PITC, and v o l a t i l e by-products were removed i n t h i s way. b. Cleavage of Phenylthiocarbamyl-Peptide or -Protein Anhydrous t r i f l u o r o a c e t i c acid (50 u l i t r e s for pep-tides, 75 u l i t r e s for protein) was added to the above dried residues. The tubes were flushed with N 2, sealed with Para-f i l m , and incubated at 45 C for 10 min. TFA was then removed by drying the samples again i n vacuo, as i n section M(4)a above. c. Removal of Diphenylthiourea (DPTU) Dried peptide residue was dissolved i n 1 ml of water-saturated ethyl acetate. Ethyl acetate-saturated water (0.25 ml) was added, mixed vigorously with a Vortex mixer, and the tubes centrifuged. The supernatant was c a r e f u l l y pipetted of f and the aqueous phase was washed twice more with 1 ml a l i -quots of the ethyl acetate. The aqueous samples were then dried i n vacuo at 60 C, and the peptide residues taken up i n 59 2 00 u l i t r e s of 50% pyridine. Samples were removed for dan-sy l a t i o n (see section M(l) above) and the degradation cycle repeated (see section M(4)a above). Dried protein residue was washed free of DPTU accord-ing to the method of Percy and Buchwald (64). The residue was washed twice with 1-ml portions of 95% ethanol to make i t less adherent to the walls of the tube. After drying, the residue was broken up f i n e l y •inE.i2-..ml:. o'f.lch'16j3©.£obm?.benzene (1:1, v/v) with a t h i n glass s t i r r i n g rod, and washed three times with t h i s solvent. The protein was washed again and then suspended i n 0.4 ml of deionized water. After three extractions with 2-ml aliquots of butyl acetate, and drying i n vacuo, the sample was taken up i n 500 u l i t r e s of 50% aqu-eous pyridine (or 50% aqueous pyridine made 10% i n N-ethyl-morpholine). A sample was removed for dansylation (see sec-t i o n M(2) above) and the degradation cycle repeated (see sec-t i o n M(4)a above). CHAPTER III RESULTS 1. CHARACTERIZATION STUDIES A. Preparative Work At the s t a r t of t h i s . i n v e s t i g a t i o n in, 1970, only a few procedures for the preparation of ferredoxin from leaves were available i n the l i t e r a t u r e . The basic method of acetone fr a c t i o n a t i o n of an aqueous leaf extract (65) had been mod-i f i e d to purify ferredoxin from some plants I.e.g. spinach C 4 6 , 66, 67, 68, 69), parsley (70, 71), Leuoaena (72), and taro (73)1. A r a d i c a l l y d i f f e r e n t procedure which involved exhaustive extraction of green cotyledons with carbon t e t r a -chloride-hexane (.2:1, v/v) and various treatments of the subsequent residue had been used for the i s o l a t i o n of cotton ferredoxin (74). However, the above methods were i n i t i a l l y avoided because t h e i r reported y i e l d s were,, not as large as those needed for sequence studies, or because of.the d i s -comfort and health hazards involved when working with orga-nic solvents i n an unventilated 4 C cold room. In contrast, Keresztes-Nagy and Margoliash had pre-pared the protein from a l f a l f a by a method which did not i n -volve organic solvents: the cheesecloth f i l t r a t e of an aqueous homogenate of a l f a l f a stems and leaves was d i r e c t l y passed . through a bed of ion-exchange r e s i n ; the r e s i n was washed, the adsorbed ferredoxin was then eluted, and the protein was 60 61 further p u r i f i e d by p r e c i p i t a t i o n and several chromatographic steps (33). However, when thi s method was attempted with Sambueus leaves, numerous d i f f i c u l t i e s were encountered. More than the reported amount of T r i s was needed to buffer the i n i t i a l extract at pH 7.5 (3.1 g of T r i s per 1.3 l i t r e H 20 instead of 1.0 g). Four layers of cheesecloth instead of only two were necessary to contain the thick homogenate when being squeezed. The extract from even one l o t of leaves, when f i l -tered through cheesecloth, did not pass through a 16 (diam) x 3 cm bed of DEAE-cellulose with the reported flow rate. In fac t , large amounts of chlorophyllous slime i n the solution interfered with the DEAE-cellulose f i l t r a t i o n (even when suc-t i o n was applied) to such a degree that flow through the bed ceased. A modification was then sought which would quickly remove p a r t i c u l a t e and l i p i d - l i k e residues p r i o r to DEAE-ce l l u l o s e f i l t r a t i o n . After f i l t r a t i o n through cheesecloth, continuous centrifugation of the extract using a dairy cream separator was slow and i n e f f i c i e n t because of the voluminous p r e c i p i t a t e . Also, p r i o r f i l t r a t i o n of the cheesecloth f i l -t r a te on a Buchner funnel with C e l i t e f i l t e r a i d, according to Lee, Travis, and Black (75), proved time-consuming and ex-pensive due to the large amount of C e l i t e needed to adsorb most of the i n t e r f e r i n g material. ' Because of these r e s u l t s , further attempts at preparation of ferredoxin from Sambueus leaves did not involve the d i r e c t f i l t r a t i o n of the aqueous extract through ion-exchange r e s i n . Instead, batch methods developed more recently were t r i e d (34, 77). Leaves (5 kg) were homogenized i n T r i s buffer contain-62 ing 1% Tween 80 (76). The homogenate was squeezed through cheesecloth and a 200 ml batch of w e l l - s e t t l e d DEAE-cellulose was added to the 12 l i t r e s of f i l t r a t e (34, 77). After s t i r -r ing for 1 hr the re s i n was allowed to s e t t l e and the super-natant was siphoned o f f . The r e s i n was c o l l e c t e d and repeat-edly washed with buffer by batch technique using a 1 l i t r e centrifuge. Further washing of the re s i n i n a column and el u t i o n of adsorbed protein with 1 M NaCl i n T r i s buffer was also slow because of poor flow rate properties of the c e l l u l o s e at that stage of the preparation. The ferredoxin-containing solution was then treated with ammonium sulphate and the pre-c i p i t a t e was removed by centrifugation. Preparation of the supernatant (ca. 2.5 l i t r e s ) for d i a l y s i s ("34") was cumbersome because of the large volume involved. The dialyzed ferredo-xin solution was concentrated and further p u r i f i e d by DEAE-ce l l u l o s e chromatography (34). Amino acid analysis of the product indicated a very low y i e l d (ca. 5 rag/kg leaf material), possibly due to degradative losses during the long extraction period. The i n i t i a l stages of aqueous extraction procedures were slow p a r t l y because i t appeared that the wax content of Sambuous leaves i n t e r f e r e d with DEAE-cellulose chromatography. This suggested that completely aqueous methods would not a l -low optimal rates of ferredoxin extraction. Therefore, e f f o r t s turned to the use of acetone i n the homogenizing medium. I t was thought that the resultant decrease i n the surface ten-sion of the extracts would increase the rate of treatment with DEAE-cellulose by improving flow rate properties. The 63 method of Crawford and Jensen, which described the i s o l a t i o n of ferredoxin from corn leaves, seemed promising (35). When Sambueus leaves were homogenized i n buffered 50% acetone-water, f i l t e r e d through cheesecloth, and c e n t r i f u -ged, the supernatant extract passed through a bed of DEAE-ce l l u l o s e very rapidly {.see Chapter I I , section 2 B(3)]. Also, ferredoxin, unlike many leaf proteins, i s soluble i n 50% acetone, so the centrifugation step was an e f f i c i e n t method of eliminating unwanted p r e c i p i t a t e and pa r t i c u l a t e matter. Although the procedure described by Crawford and Jensen worked well with Sambueus leaves, the y i e l d of pure protein from one such extraction (10 mg/400 g leaves) was considered inconveniently low for purposes of accumulating large enough amounts of material for sequence studies. Therefore, large-scale preparations of ferredoxin using mod-i f i c a t i o n s of the basic procedure of Crawford and Jensen were attempted. However, i f volumes of solutions were increased too much, those steps involving column chromatography became unworkable. The method outlined i n Chapter I I , section 2 B(3), u t i l i z i n g common laboratory apparatus, proved.to be the most e f f i c i e n t adaptation. The extraction procedure (to the end of p u r i f i c a t i o n step d) was accomplished i n 48 hr. The y i e l d was 18-20 mg of freeze-dried ferredoxin from 800 g fresh weight of leaves (22.5-25.0 mg/kg leaf material). The reported yi e l d s of ferredoxin from leaves of other plants are shown i n Table I. The large d i f f e r e n c e . i n the yiel d s of ferredoxin from plants picked at various times of the year, which was observed with a l f a l f a (33), was not noticed with Sambueus. TABLE I YIELD OF FERREDOXIN FROM.SAMBUCUS AND COMPARISON TO YIELDS FROM OTHER PLANTS Y i e l d Source (mg/kg fresh wt) Sambueus leaves 222 55-25.0 Equisetum (78) 2.0-5.0 Leuoaena leaves (72) 20.0 Spinacea leaves 22.0 Medicago stems and leaves (33) 20.0-25.0 Colocassia leaves (73) 25.0 Zea leaves (35) 37.5 Gossypium cotyledons (74) 200.0 a: Fresh weight not reported. Calculation based on y i e l d of ferredoxin from l y o p h i l i z e d , solvent-extracted dried cotyledons. 64 However, one seasonal difference was pronounced. During chromatography on the 2.5 x 45 cm column of DEAE-cellulose (see Chapter, section 2 B(3), p u r i f i c a t i o n step c ) , extracts of leaf shoots and very young leaves obtained i n early spring showed more varied and intense polyphenol-like colouration than extracts of late spring and summer leaves. The migration of the ferredoxin-containing yellow red band down the column was therefore more d i f f i c u l t to see. In most preparations though, ferredoxin-containing fractions c o l -lected from the 2.5 x 45 cm DEAE-cellulose column could be i d e n t i f i e d by t h e i r UV spectra. An absorption peak or shoul-der at 330 nm and asmall peaks at 410-414 nm and 464-466 nm were c h a r a c t e r i s t i c of the protein. During the f i n a l preparative step - gel chromatography of the concentrated ferredoxin-containing solution through a 4.7 x 100 cm column of Sephadex G-75 - ferredoxin migrated ahead of two slower moving fluorescent bands. A t y p i c a l elution p r o f i l e i s shown i n Figure 4. Crawford and Jensen found only one fluorescent band at t h i s stage during prepar-ation of corn ferredoxin (35). The fluorescent contaminants gave a negative t e s t for flavonoids and appeared to be poly-phenol condensation products. The ferredoxin band c o l l e c t e d from the Sephadex G-75 column was pure, as indicated bye disc electrophoresis and other c r i t e r i a (see t h i s chapter, section 1 D) . i 1 1 1 1 1 1 1 1— : i 1 1 1 r Ferredoxin Fraction Number ( 2 0 ml ) F i g . 4 Sephadex G-7 5 chromatography of f e r r e d o x i n - c o n t a i n i n g s o l u t i o n p r e v i o u s l y f r a c t i o n a t e d on DEAE-cellulose. The sample was a p p l i e d to a 4.7 x 100 cm column e q u i l i b r a t e d , w i t h 10 mM T r i s - H C l , 0.2 M NaCl, pH 7.5. F r a c t i o n s were c o l l e c t e d at 60 ml/hr. 67 B. Spectral Properties The UV and v i s i b l e absorption of p u r i f i e d Sambucus ferredoxin was measured on a Unicam SP1800 dual beam record-ing spectrophotometer. Spectra were recorded immediately after p u r i f i c a t i o n . Samples were either taken d i r e c t l y from the Sephadex G-75 column eluate, or af t e r the eluate was concentrated (see Chapter I I , section 2 B(3), p u r i f i c a t i o n step e) . The absorption spectrum i s shown i n Figure 5, (Curve A). Absorption maxima occur at 277, 331, 423, and 466 nm. These average positions are based on the spectra of eight d i f f e r e n t preparations of Sambucus ferredoxin and have a range of ±1 nm. Table II compares the absorption maxima of other plant ferredoxins with.those of Sambucus.. The r a t i o s (."critical ratios") of the absorbances at 466, 423, and 331 nm to that at 277 nm are 0.44, 0.49, and 0.67. Table III presents these values together with the absorbance r a t i o s of other f e r -redoxins. The molar extinction c o e f f i c i e n t at 423 nm ( £ 4 2 3 ) / calculated from a molecular weight of 11,000, varied from - 1 - 1 10.8 to 12.9 mM xcm , when the concentration of the ferredo-xin solutions was determined on a dry weight basis. When the concentration was determined by d i r e c t iron analysis, assuming . . . _ i 2 atoms of iron per molecule of ferredoxin, £1* 2 3 was 9.1 mM cm 1 , The molar extinction c o e f f i c i e n t s of plant ferredoxins are given i n Table IV. C. Electron-Transfer A c t i v i t y The p u r i f i e d protein exhibited ferredoxin a c t i v i t y by 0 I • ' ' ' i i i i ' • • i • • 2 5 0 310 3 7 0 *»30 4 9 0 5 5 0 WAVELENGTH (nm) F i g . 5 Absorption spectrum of native and deteriorated ferredoxin from Sambucus racemosa. Curve A i s the spectrum of a ferredoxin preparation with the highest absorbance r a t i o (423/277 nm) of 0.49. Ferredoxin (ca. 1 mg/ml) was i n 0.1 M Tris-HCl, 0.8 M NaCl, 1 mM 3-mercaptoethanol, pH 7.5. Curve B i s the spectrum of apoferredoxin. Ferredoxin was desalted on Sephadex G-25 with d i s t i l l e d water. TABLE I I ABSORPTION MAXIMA OF PLANT FERREDOXINS Source of Ferredoxin Wavelength (run) of Absorption Maxima (M) Mi M2 M3 M,, Angiosperms Sambueus 277 Leuoaena (72) 277 Spinacea (79) 274 Medicago (33) 277 Colooassia (73) 277 Zea (35) 277 Gossypium (74) 280 Fern Polystichum C80) 276 Primitive Vascular Plant Equisetum (78) 276 Green Algae Soenedesmus (12) 276 Euglena (81) 276 Blue-green Algae Nostoa (82) 276 Microcystis (83) 276 331 325 325 331 330 330 325 330 330 330 328 331 330 423 420 420 422 420 423 419 420 421 421 422 423 422 466 463 463 465 465 463 460 465 465 464 465 470 464 69 TABLE III CRITICAL ABSORBANCE RATIOS OF PLANT FERREDOXINS Source of Ferredoxin Ratio of absorbance at different, maxima (M) to that at Mi Ccf. T a b l e l l l ) Mi Mi Ms Angiosperms Sambueus Leucaena (72) . Medieago (33) Colooassia (73) Zea (35) Green Algae Seenedesmus (45) Euglena (81) Blue-green Alga Nostoe (82) 0.44 0.43 0.43 0.39 0.43 nr 0.60 nr 0.49 0.49 0.48 0.43 0.48 0.65 0.68 0.57 0. 67 0.65 0.65 0. 64 0.60 0.88 0.87 nr nr: Not included i n reference. 70 TABLE IV MOLAR EXTINCTION COEFFICIENTS OF PLANT FERREDOXINS Source of f Ferredoxin Wavelength (nm) CmM~;icm~1) Angiosperms Sambucus 423 9.I a Spinacea (12) 420 9.7 Medicago (12) 422 9.1 Colocassia (73) 420 9.7 Zea (35) 423 10.0 Gossypium (74) 419 7.6 Petroselinum (84) 422 9.2 Primitive Vascular Plant Equi-setum (78) 421 8.8 Blue-green Alga Spirulina (85) 420 9.7 a: Value calculated from absorbancy measure-ment and d i r e c t iron analysis of the same ferredoxin solution with an absorbance r a t i o (423 nm/277 nm) of 0.49. 71 72 mediating the photoreduction of NADP by spinach chloroplasts. Figure 6; shows the resultant increase i n the absorption of the reaction mixture at 340 nm due to the formation of NADPH2. Sambucus ferredoxin sustained an i n i t i a l reduction rate of 86 umoles NADP per mg chlorophyll per hour. D. Homogeneity The homogeneity of ferredoxin preparations was assessed by disc electrophoresis. In 7.7% polyacrylamide gels, f e r -redoxin migrated together with the bromophenol blue front marker dye as a single, t h i n , f a s t moving, coloured band. A l l samples examined showed no evidence of contamination. Figure depicts the t y p i c a l pattern of stained protein i n the gels. A useful index of purity i s the comparison of the l i g h t e xtinction of iron-sulphur proteins at c h a r a c t e r i s t i c wavelengths (absorbance maxima). Absorbance r a t i o s have almost always been used as one c r i t e r i o n of homogeneity i n studies involving ferredoxin (4, 33). In Sambucus ferredoxin preparations, the 423 nm to 277 nm absorbance r a t i o varied from 0.45 to 0.49. This i s comparable to the r a t i o s of other pure ferredoxins (Table) JI^^^^ of purity. Also, s i m i l a r i t y of the spectrum (Figure 5 ), absor-ption maxima (Table I I ) , and molar extinction c o e f f i c i e n t , (Table IV.)ib6f Sambucus ferredoxin to the corresponding spec-t r a l properties of other p u r i f i e d ferredoxins was used to judge the protein preparations homogeneous. 1-4 TIME (min) F i g . 6 Sambucus ferredoxin-mediated photoreduction of NADP. The reaction mixture contained 2 umole of NADP, chloroplast suspension with 0.1 mg of chlor o p h y l l , 0.1 mg of ferredoxin, and 0.25 M T r i s , 0.5 M s o r b i t o l , 5 mM (3-mercaptoethanol at pH 7.5, i n a f i n a l volume of 3.0 ml. The blank cuvette contained the same components except for ferredoxin. The ferredoxin sample had an absorbance r a t i o (423/277 nm) of 0.44. See Chapter I I , section 2 D for conditions of assay. — i 74 ( - ) r - S H ( + ) A B F i g . 7 Electrophoretic pattern of Sambueus ferredoxin. L: large-pore spacer g e l . S: 7.7% polyacrylamide small-pore separating g e l . A: 60 yg of ferredoxin (with a 423/277 nm absorbance r a t i o of 0.268) was applied to spacer g e l . B: 200 yg of ferredoxin (423/277 nm r a t i o of 0.47). In a l l gels, ferredoxin moved with the electrophoretic front. See Chapter II, section 2 E for conditions of electrophoresis. 75 The f i n a l step for p u r i f i c a t i o n of ferredoxin was gel f i l t r a -t i o n through a 4.7 x 100 cm column of Sephadex G-75 [see Chapter I I , section 2 B(3)J. The elution p r o f i l e indicated a clean sharp peak corresponding to the ferredoxin band (Figure 4) . Symmetry of the peak suggested the presence of a single component. Other close peaks, shoulders, or skew-ness, i n d i c a t i v e of possible contamination, were absent. The v i s i b l e l i g h t absorbance at 420 nm, due to the iron chromophore (33), coincided with the UV l i g h t absorbance at 280 nm, due to protein aromatic amino acids. This fur-ther showed that the peak represented only ferredoxin. Electrophoresis of fluorescamine-labeled samples on polyacrylamide gels i n the prescence of SDS was performed to estimate the molecular weight, of Sambueus. ferredoxin (see t h i s chapter, section F). In a l l gels, only one t h i n fluorescent band was v i s i b l e . On no occasion during the sequence, studies was there any evidence of heterogeneity i n the ferredoxin preparations. The NH 2-terminal determinations performed on the whole pro-t e i n as well as on peptides always showed the presence of only one amino terminus. Furthermore, high stoichiometry among amino acid re-sidues was evident i n the amino acid analyses. E. Amino Acid Composition The results of the amino acid analyses of Sambueus f e r -redoxin are given i n Tables V, VI, VII, and VIII. Three sam-ples of protein were hydrolyzed with 6 N HC1 i n vacuo for 19, TABLE y AMINO ACID ANALYSES OF DESICCATED HYDROLYZATES OF SAMBUCUS FERREDOXIN.9, (nmole amino acid/mg protein) Values obtained after hydrolysis f o r : Amino acid 19 hr 38 hr 62 hr Lysine 282.9 C 304.8 309.9 309.9 Hist i d i n e 72.6 119.0 127.8C 127.8 Arginine 58.1 69.7 73.4 73.4 Aspartic acid 650.4 752.4 771.8 771.8 Threonine 262.2 336.2 ., 337.9 C 337.9 Serine 415.9 472.7 494.9° 494.9 Glutamic acid 1002.8 1150.7 1144.2 1150.7 Proline 257.9 355.4 338.1 355.4 Glycine 428.9 475.3 498.5° 498.5 Alanine 379.2 429.9 445.9 445.9 Half cystine 176. 3 266.4C 262.1 266.4 Valine 247.1 478.2° 495.6 495.6 Methionine 0 0 0 0 Isoleucine 222.5 346.3 C 355.2 355.2 Leucine 326.4 440.3 454.1 454.1 Tyrosine 187.8 235.3 C 248.6° 248.6 Phenylalanine 117. 3 144.4 148.2 148.2 a: See Chapter I I , section 2 G(l) for d e t a i l s of ana-l y s i s procedures. b: Maximum value. c: Higher than corresponding value from undesiccated hydrolyzate (Table VI). Best b estimate 76 TABLE YI AMINO ACID ANALYSES. OF UNDESICCATED HYDRQLY ZATES OF SAMBUCUS FERREDOXIN^ Cnmole amino acid/mg protein) Amino acid Values obtained after hydrolysis f o r : 19 hr 38 hr 62 hr Best k Percent estimate increase Lysine H i s t i d i n e Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine P heny1a1anine Average Percent 260.5 79.3 66.6 743. 8 291.6 470.1 1071.1 316.8 435.1 404.4 212.5 284.8 0 252.5 365.8 201.8 153.8 Increase d 308.2 139.5 73.8 812.6 355. 3 551.9 1206.7 384.5 485.8 448.7 261.5C 468.6C 0 342. 8C 446. 6 232.8C 165.9 391, 106, 75. 838. 296. 481, 1249. 439, 486. 465. 277, 619, 0 381. 461. 239. 173. 5 391.5 4 d 139.5 4 75.4 8 838.8 3^353355.3 2d551551.9 2 1249.2 439.8 486.6 465.3 277. 2 619.7 0 381.7 461.6 239.1 173.8 7.0 1.8 7.2 10.1 0.7 8.6 7.0 19.9 0.3 5.0 6.6 12.5 5.7 4.4 0.3 20.4 7.3 a: Hydrolyzates were not desiccated according to the method of Robel (40). See Chapter I I , section 2 G(l) for de-t a i l s of the analysis procedures.. b: Maximum value.'" c: The increase i n t o t a l amino acid recovered from the 3 undried hydrolyzates (this Table), expressed as a per-centage of t o t a l amino acid recovered from the 3 dried hydro-lyzates (see Table V) . d: Lower than corresponding value from dried hydro-lyzates (Table) , 77 TABLE yiX AMINO ACID COMPOSITION OF SAMBUCUS FERREDOXIN Amino . Average of Residues per Nearest acid best estimates molecule integer Lysine 350. 7 5.00 5 Hist i d i n e 133.7 2. 00 2 Arginine 74.4 1.00 1 Aspartic acid 805.3 11.48 11 Threonine 346.6 5.00 5 Serine 523.4 7.46 7 Glutamic acid 1200.0 17.11 17 Proline 397.6 5.67 6 Glycine 492.6 7.00 7 Alanine 455.6 6.50 6-7 Cysteine 0 271.8 3.88 4 Valine 557. 7 8.00 8 Methionine 0 0 0 Isoleucine 368.5 5.25 5 Leucine 457.9 6.53 7 Tyrosine 243.9 3.48 3 Phenylalanine 161.0 2.30 2 Tryptophan 9 2 . l d 1.31 1 Total residues 97-98 a: Best estimates (nmole amino acid/mg protein) from 'de-siccated and undesiccated hydrolyzates are shown i n Table V and TaBl-e VI, respectively. *:r b: Assuming 1 arginine, 2 h i s t i d i n e , 5 l y s i n e , 5 threo-nine, 7 glycine, and 8 valine per molecule of ferredoxin. c: Also determined as AECys. Analysis of undried 24 hr hydrolyzate of AECys-ferredoxin showed 229.1 nmole/mg protein. d: Determined according to the method of L i u (43). See Chapter I I , section 2 G(2) for d e t a i l s of analysis. 78 TABLE VIII AMINO ACID COMPOSITION OF PLANT FERREDOXINS Lys His Arg Trp Asx Thr Ser Glx Pro Gly Ala Cys Val Met H e Leu Tyr Phe T o t a l Sambucus 5 2 1 1 11 5 7 17 6 7 6-7 4 8 0 5 7 3 2 97-98 Leucaena (72) 5 1 2 1 11 4 7 16 5 6 7 5 6 0 4 10 3 3 96 Medicago 9 (86) 5 2 1 1 9 6 8 16 3 7 9 5 9 0 4 6 4 2 97 Spinacea 3 (50) 4 1 1 1 13 8 7 13 4 6 9 5 7 0 4 8 4 2 97 Zea mays (35) 3 2 1 1 13 5 8 14 4 8 8 4 10 0 5 8 5 1 100 a Colocassia (49) 5 1 1 1 10 6 8 15 4 9 6 5 10 0 4 6 4 2 97 Polystichum (80) 4 2 1 0 14 6 8 9 5 9 7 5 5 2 6 7 3 4 97 Equisetum (78) 4 1 l 1 0 9 7 8 16 4 9 6 4 6 1 5 8 2 4 95 a Scenedesmus (87) 4 1 1 0 12 10 8 10 4 7 10 6 5 1 3 7 4 3 96 M i c r o c y s t i s (83) 3 1 1 0 13 7 6 13 4 12 9 5 4 1 6 9 3 1 98 a . Amino a c i d sequences known. <D \ 80 38, and 62 hr. After the tubes were opened the hydrolyzates were desiccated, dissolved i n 0.2 N sodium c i t r a t e buffer, and then analyzed according to standard procedure. The re-covery, of each amino acid from these three hydrolyzates i s shown i n Table V. Three duplicate hydrolyzates were not dried; instead, they were d i r e c t l y d i l u t e d with 0.2 N sodium c i t r a t e buffer [see Chapter I I , section 2 G(1)J. Table VI presents the r e s u l t s from these l a t t e r samples. Comparison of the 48 pairs of corresponding values (16 amino acids x 3 d i f f e r e n t hydrolysis times) i n Tables V and VI shows that only ten values were higher i n the desiccated hydrolyzates. In most cases, higher values were recorded for the undesiccated hydrolyzates. The t o t a l difference between the recovery of each amino acid from the two types of hydrolyzates i s expressed as a percent-age i n Table VI. Every amino acid was recovered i n a higher average y i e l d from the undesiccated hydrolyzates than from those hydrolyzates which were dried before analysis, and the increase varied with each amino acid from 0.3% (tyrosine and glycine) to 20.4% (phenylalanine). Aspartic acid (10.1%), valine (12.5%), proline (19.9%), and phenylalanine (20.4%) showed the highest increases. The average increase i n reco-very of amino acids from hydrolyzates not dried before analy-s i s was 7.3%. Methionine was not detected. A l l other amino acids, including serine and threonine, were recovered i n larger am-ounts from the 38 hr hydrolyzates than from the 19 hr hydro-lyzates. Most amino acids yielded t h e i r highest values a f t e r 81 62 hours of hydrolysis. For t h i s reason the results from d i f -ferent hydrolysis times were not averaged; instead, the ma-ximum value of each amino acid was assumed to be the best es-timate of i t s actual occurence i n the native protein. The best estimate values from desiccated hydrolyzates (Table V) and undesiccated hydrolyzates (Table VI) were averaged. The average values are shown i n Table VII, together with the re-s u l t of the tryptophan analysis which was performed by the method of L i u (43). The average value for cysteine was higher than the value obtained from analysis of S - 6-aminoethylcys-teinylferredoxin; and therefore the average value probably approximates the maximum occurence of cysteine i n the protein. The amino acid composition was calculated with arginine nor-malized to one residue per molecule of ferredoxin, h i s t i d i n e normalized to two residues, ly s i n e normalized to f i v e , threo-nine to f i v e , glycine to seven, and valine to eight. Table VII gives the resultant amino acid composition. The c a l c u l a -tions show that al'anibne may occur as s i x or seven residues per molecule of protein. The calculations also show four other amino acids (aspartic acid, serine, leucine, and tyrosine) with poor i n t e g r a l stoichiometry. Therefore, the actual t o t a l number of residues may vary from 97 to 102. But i f homology with other plant ferredoxins i s assumed (see Table VIII for comparison), 97 or 98 residues is. probably correct. The amino acid composition of Sambueus ferredoxin presented i n t h i s thesis includes aspartic acid, serine, and tyrosine rounded o f f to the nearest integer. 82 The contents of various amino acids of Sambucus f e r -redoxin resemble those of algae and other higher plant f e r -redoxins (Table VIII). The numbers of l y s i n e , tryptophan, aspartic acid, serine, alanine, and tyrosine residues are i d e n t i c a l to Leucaena ferredoxin. The content of l y s i n e , h i s -t i d i n e , arginine., tryptophan, glycine, and phenylalanine are i d e n t i c a l to Medicago ferredoxin. The t o t a l basic amino acid content 19 residues) i s i d e n t i c a l to that of Leucaena 3 Medi-cago, and Cyperus (75). The r e l a t i v e proportions of aspartic acid and glutamic acid are remarkably si m i l a r to those found i n other plant ferredoxins: Leucaena 3 Medicago Co locassia 3 Gossypium (74), Equisetum, .@&p.e&ft&d<k$S)>gaai&&8@ladophora (88). The occurence of 6 proline and 17 glutamic acid residues i n Sambucus ferredoxin has not been reported for other ferredo-xins (5, 20). F. Molecular Weight The minimum value for the molecular weight of Sambucus ferredoxin calculated from the aminoo.acid composition reported i n Table VII i s 10,610 daltons. Including the two atoms of iron and two atoms of inorganic sulphur i n the active centre (see t h i s chapter, section 1 G), the minimum molecular weight i s 10,786. The molecular weight was also determined from the e l -ectrophoretic mobility of fluorescamine-labeled ferredoxin i n SDS gels r e l a t i v e to molecular weight standards. The protein 83 standards used, were .ovalbumin.. (45,000) , chymotrypsinogen (25,000)., myoglobin (17,800), lysozyme (14,300), and ribonu-clease (12,700). Figure 8 shows the res u l t s of a t y p i c a l electrophoretic run. Values for ferredoxin repeatedly ranged from 1.02 x 10 4 to 1.12 x 10 4 daltons. The average molecular weight of ferredoxin from t h i s method i s 10,700 ± 500 daltons. The values from the two d i f f e r e n t methods are consis- • tent and give an average molecular weight of Sambueus ferred-oxin of 10,740 ± 600. This i s si m i l a r to the values reported for other plant ferredoxins. For example, the formula mo-lecular weights of Leueaena (72) and Spinaeea (50) f e r r e -doxins are 10,770 and 10,646, respectively. G. Iron Content The iron content of ferredoxin was determined by the o-phenanthroline colorimetric. method. Table IX shows the results from three t y p i c a l analyses. The values suggest that two iron atoms are present i n one molecule of Sambueus ferredoxin, as i n a l l well characterized plant ferredoxins (5). From a preliminary analysis, the content of inorganic l a b i l e sulphide seemed to be 2 g atom per molecule. Together with the r e s u l t s of the iron analysis, t h i s suggests the active centre of Sambueus ferredoxin to be the same as i n other plant ferredoxins. 84 MIGRATION DISTANCE (cm) F i g . 8 SDS e l e c t r o p h o r e s i s of fluorescamine-labeled f e r r e d o x i n and molecular weight standards on 10% polyacrylamide g e l s . P l o t of m i g r a t i o n d i s t a n c e versus logarithm of molecular weight. Distances are normalized to a bromophenol blue f r o n t m i g r a t i o n d i s t a n c e of 5 cm. A b b r e v i a t i o n s : O, ovalbumin; M, myoglobin; C, chymotrypsinogen; L, lysozyme; R, rib o n u c l e a s e ; F 1 , F 2 , samples of Sambueus f e r r e d o x i n from two d i f f e r e n t pre-p a r a t i o n s . TABLE IX IRON CONTENT OF SAMBUCUS FERREDOXIN P r o t e i n 3 (umole) Iron Ratio - Nearest (umole) (g atom/molecule) integer 0.0154 0.0173 0. 0224 0.0347 0.0291 0.0546 2.256 1.682 2.438 2 2 2 a: Determined on dry weight basis, assuming molecular weight of 10,700. Samples were from d i f -ferent preparations. 85 2.. • PRELIMINARY SEQUENCE STUDIES Trie NH 2-terminal amino acid residue of Sambueus f e r -redoxin was determined by the dansyl method (58). Protein was denatured by heating i n 1% SDS and buffered with N-ethyl-morpholine. Dansyl chloride, dissolved i n dimethylformamide, was then added to the protein and allowed to react. When re-agent grade dimethylformamide was d i s t i l l e d and used as the solvent for the dansyl chloride, no dansylated products were detected aft e r hydrolysis of the protein. I t seemed that even small traces of water in t e r f e r e d with the l a b e l l i n g of the protein. This problem was solved by the use of anhydrous s i l y l a t i o n grade dimethylformamide as s p e c i f i e d i n Chapter I I , section 1 B. After reaction, the dansylated ferredoxin was precipitated with acetone and washed free of excess reagents. If the labeled protein was washed with 80% acetone as suggested by Gray (58), the ferredoxin redissolved and only p r e c i p i -tated again on long standing at 4 C. Instead, 90% acetone was used to wash the dansylated ferredoxin which, i n t h i s case, remained prec i p i t a t e d as tiny f l o c c u l i . The dansyl amino acid l i b e r a t e d from the dansyl de-r i v a t i v e of native ferredoxin upon hydrolysis was i d e n t i f i e d as dansyl alanine by thin-layer chromatography. e-Dansyl ly s i n e was also detected on the polyamide sheet due to the content of f i v e l y s i n e residues i n the protein. Attempts at dansyl-ating the NHi-terminus of AECys-ferredoxin f a i l e d . Only e-dansyl l y s i n e was detected. The apparent reason was the i n s o l u b i l i t y of the AECys-derivative of Sambueus ferredoxin 86 87 Csee below) which, did not dissolve even i n the 1% SDS-N-ethyl-morpholine^dimethylformamide (2:2:3 by volume) dansyl reac-t i o n medium; and that the NH 2-terminus was therefore not ex-posed to the dansyl chloride and so unable to react with i t . After one cycle of Edman degradation performed on native ferredoxin, the second amino acid residue was i d e n t i f i e d as dansyl threonine. Although AECys-ferredoxin was insoluble -in the coupling buffer, i t was ca r r i e d through one cycle of degradation nevertheless, with the hope that during the sub-sequent dansylation procedure, the NH 2-terminus of the poly-peptide chain would be exposed to and react with the dansyl chloride. However, no new dansyl amino acid was detected, apparently for the same reason as stated above. These re s u l t s suggest that the amino-terminal sequence of native Sambucus ferredoxin i s Ala-Thr. Apoferredoxin, prepared by t r i c h l o r o a c e t i c acid t r e a t -ment, appeared to be well hydrolyzed by thermolysin under the digestion conditions described i n Chapter I I , section 2 J ( l ) . The thermolytic peptide mixture was fractionated by gel f i l -t r a t i o n through Sephadex G-2 5 Fine. The elu t i o n p r o f i l e i s shown i n Figure (9. On the basis of l i g h t absorbancy at 220 nm Cpeptide bonds) and 280 nm (aromatic amino acids), the column eluate was pooled into f i v e f r a c t i o n s . Two-dimensional elec-trophoresis showed that each f r a c t i o n was substantially hetero-geneous: fractions A, B, C, D, and E contained 32, 38, 22, 14, and 9 ninhydrin-positive spots respectively. The f i r s t three fractions shared a considerable number of spots with i d e n t i c a l m o b i l i t i e s . Six thermolytic peptides (Th-1 to Th-6) were i s o -EFFLUENT VOLUME ( ml ) F i g . 9 Elution pattern of peptides from the thermolytic digest of TCA-treated ferredoxin chromatographed on a column of Sephadex G-25 Fine (1.4 x 97 cm). Letters r e f e r to the fractions pooled which are indicated by the v e r t i c a l l i n e s . Thermolytic peptides studied (Th-1 to Th-6) were isolated from the pooled f r a c t i o n s indicated. See Chapter I I , sections 2 J ( l ) and 2 K for experimental d e t a i l s . g 89 lated and p a r t i a l l y studied. The. amino acid compositions of three peptides (Th-1, -3, and -4) are presented i n Table X. Peptide Th-1: Leu-Asx (Asx, Glx, Glx)'. This was an a c i d i c peptide i s o l a t e d from Fraction A. I t stained red with ninhydrin. Mobility Cm) with respect to dansyl arginine was 0.39 at pH 6.5 and 0.4 5 at pH 1.9. Having an. approximate molecular weight of 600, these mobility c h a r a c t e r i s t i c s i n -dicate that the peptide has a net charge of +1 at pH 1.9., according to Bailey and Ramshaw (89). Two dansyl-Edman steps indicated the amino-terminal sequence to be Leu-Asx. Peptide Th-2: neutral peptide from Fraction B, stained red, m 6 . 5 - = 0.90, m i . 9 = 0.74. Two dansyl-Edman steps i n -dicated the amino-terminal sequence to be Leu-Gly. Peptide Th-3: Leu-Thr (Ala). A neutral peptide from Fraction C, stained red, m 6 .5 = 0..9I, m i . 9 = 0.65. Amino-u.. *t J. terminal sequence was Leu-Thr. The complete sequence must therefore be Leu-Thr-Ala. Peptide Th-4: Ile-Asx (Glx, Gly, Trp, V a l ) . An a c i d i c peptide from Fraction C, m6.s = 0.17, itii. 9 = 0.39. Fluores-cence i n UV l i g h t indicated the presence possibly of ferredoxin's single tryptophan residue. I t stained l i g h t red with ninhydrin but became dark red overnight i n d i c a t i n g valine or isoleucine as the NH 2-terminal residue (55). The molecular weight of the peptide and i t s mobility at pH 1.9 with.respect to dansyl-arginine indicate a net charge of +1. Two dansyl-Edman steps indicated the amino-terminal sequence to be Ile-Asx. Peptide Th-5: basic peptide from Fraction E, stained TABLE X AMINO ACID COMPOSITION OF THERMOLYTIC PEPTIDES 3 OF TCA-TREATED FERREDOXIN The composition of each peptide i s given as the molar r a t i o s of the amino acids, calculated with-out, correction for destruction during 6 N HC1 hydrolysis for 24 hr. The i n t e g r a l values of major constituents are given i n parentheses. Amino acid Th-•1 Th- 3 Th- 4 Lysine 0. . 03 Aspartic acid 2. .15 (2) 0. .14 1. .27 CD Threonine 0. .73 (1) Serine 0. .06 Glutamic acid 2. .18 C2) 1. .27 CD Glycine 0. .21 1. .19 (1) Alanine 1. .13 CD Valine 0. .18 1. .00 CD Isoleucine 1. .06 CD Leucine 1. .00 (1) 1. .00 CD Total,! Residues 5 3 5 a: See Chapter I I , sections 2 J (1), 2 K, and 2 L for experimental d e t a i l s . P u r i f i e d pep-tides were eluted from paper and divided into two equal halves for amino acid analysis and dansyl-Edman sequencing. 90 91 red, ni6 • 5 = 1.60, jtij. a = 1.14. Amino-terminal sequence was Tyr—Leu. Peptide Th-6: neutral peptide from Fraction E, stained red, m 6.5 = 0.90, mi. 9 = 0.84. Amino-terminal sequence was Leu-Gly, the same as for peptide Th-2. The:increased mobi-l i t y of Th-6 at pH 1.9 as compared to that of Th-2, and i t s larger elution volume from the Sephadex G-25 column both sug-gest that peptide Th-6 may be a shorter version of the neut-r a l peptide Th-2. S - 3-aminoethylcysteiny,lferredoxin was insoluble at pH 8.2, the pH optimum of t r y p s i n . The digest.was performed, at pH 8.9 instead, for s i x hours. When the digest was c l a -r i f i e d by centrifugation the p e l l e t appeared to contain most of the o r i g i n a l protein, an i n d i c a t i o n that l i t t l e digestion had occurred. Nevertheless, the supernatant was chromato-graphed on Sephadex G-25 Fine, to obtain a.fractionation pat-tern of the t r y p t i c peptides. Figure TO shows the e l u t i o n p r o f i l e . The eluate was pooled into three f r a c t i o n s , A, B, and C, which were.shown to contain 29, 24, and 11 ninhydrin-p o s i t i v e spots respectively, by two-dimensional high voltage paper electrophoresis. Due to t h e i r low y i e l d , these tryp-t i c peptides were not studied. e c o oo CM -P ed <D o C d jo U o 09 < EFFLUENT VOLUME ( ml ) F i g . 10 E l u t i o n pattern of peptides from the t r y p t i c digest of Sambucus AECys-ferredoxin chromatographed on a column of Sephadex G-25 Fine (1.4 x 97 cm). Letters r e f e r to the fractions pooled which are indicated by the v e r t i c a l l i n e s . See Chapter I I , sections 2 J(2) and 2 K for experimental d e t a i l s . ^ CHAPTER .jy DISCUSSION A sa t i s f a c t o r y method was developed for the i s o l a t i o n and p u r i f i c a t i o n , of ferredoxin from leaves of Sambucus racemo-sa. The method, based on the techniques of Crawford and Jen-sen (35), was modified to process 800 g of leaf material at one time as rapid l y as possible. I n i t i a l l y , aqueous extraction methods (33, 34, 75, 77). were t r i e d with Sambucus leaves but these proved unsuit-able. The f i l t r a t i o n and chromatographic steps were made d i f -f i c u l t by the presence of chlorophyllous slime, and wax-like material, the content of which i n Sambucus leaves appeared to be greater than i n Medicago or Spinacea, with which the aqueous methods were developed. Others have encountered s i -milar problems. Crawford and Jensen reported that the aqueous extraction method of Keresztes-Nagy and Margoliash (33) was unsatisfactory for the i s o l a t i o n of ferredoxin from leaves of Zea mays (35). Margoliash"s group was unable to recover any ferredoxin whatsoever when large quantities of the dark a c i d i c oxidation product of phenoloxidase were present (Dr. E. Mar-goliash, personal communication). F i n a l l y , extraction of Sam-bucus leaves with 50% acetone-water allowed the prepara-tion of ferredoxin to proceed i n a fast and e f f i c i e n t manner. The extraction procedure i s described i n d e t a i l i n Chapter I I , section 2 B(3). Although i t worked well with 93 94 Sambueus- leaves./ yiburnum and. Lon%cera. leaves, f a i l e d to y i e l d any ferredoxin when thi s method was used. Howeyer, the a p p l i -c a b i l i t y of the procedure to other plant sources was not ex-tensively investigated. The y i e l d of ferredoxin (22.5 to 25.0 mg per kg leaf material) from Sambueus i s very s i m i l a r to the y i e l d s from other higher plants (Table. I ) . . Although the methods of f e r -redoxin extraction have d i f f e r e d , the reported y i e l d s from leaves of Spinaeea, Leuoaena, Colooassia, Medioago3 and Sam-bueus are a l l 20.0 to 25.0 mg. per kg leaf material. Crawford and Jensen used 20 to 35 day-old seedlings of Zea mays and obtained 37.5 mg of ferredoxin per kg of leaves (35). The y i e l d reported from Equisetum (2.0 to 5.0 mg of ferredoxin per kg leaves) i s much lower. This value may represent the actual f e r -redoxin. content i n this, group of primitive vascular plants. Such a low y i e l d from Equisetum may also be a r e s u l t of the s p e c i f i c extraction method which was applied to t h i s plant material (78). If the ferredoxin content of ferns were i n -vestigated, the r e s u l t might indicate an increasing trend from the primitive vascular plants to the Angiospermae. In t h i s respect i t i s unfortunate that i n the study of Polystichum ferredoxin, the y i e l d was not reported (80). In a recent study of ferredoxin development i n Phase-olus vulgaris, the leaves of 14 day-old e t i o l a t e d plants con-tained 1.42 to 1.76 nmol/g fresh weight (90). Assuming a mol-ecular weight of 11,000, t h i s represents 15.6 to 19.4 mg of ferredoxin per kg leaf material. During subsequent il l u m i n a -95 t i o n of the plants at high light, intensity- (2500 Lx) f o r 50 hr, the amount of ferredoxin per leaf increased approximately e i g h t - f o l d . ^ However, the weight of the leaves during t h i s per-iod was not monitored. After 50 to 70 hours the net synthesis of ferredoxin l e v e l l e d o f f . In the present case with Sambueus leaves, the yield, of ferredoxin at various developmental stages was not studied. The y i e l d of. ferredoxin from leaves of higher plants i s much greater.than the y i e l d of other proteins used i n amino acid sequence studies of plant r e l a t i o n s h i p s . Boulter and co-workers have reported y i e l d s of cytochrome o to vary from 0.2 to 0.45 mg per kg dry seeds (91). Young inflorescences of Brassiaa oleraeea and whole plants of Arum maculatum yielded 0.2 and 0.09 mg of cytochrome c per kg.fresh weight respec-t i v e l y (92). This same group has sequenced plastocyanin from Sambueus nigra and Solanum tuberosum (26, 27). The y i e l d of p u r i f i e d protein from the l a t t e r source was 3.5 mg per kg of leaves. Sambueus ferredoxin, prepared by the extraction pro-cedure presented i n t h i s thesis, was shown to be pure by disc electrophoresis i n 7.7% polyacrylamide gels at pH 9.0. The protein, due to i t s low molecular weight and high con-tent of a c i d i c amino acids, exhibited the same high anionic mobility (Figure 7) as reported for other ferredoxins (33, 34, 45, 75, 93). Electrophoresis at an additional pH le.g. pH-4.3 (75)] offers a further test of homogeneity,. but to con-serve ferredoxin material, t h i s was not performed. Keresztes-Nagy and Margoliash noted that deteriorated preparations of 96 a l f a l f a , ferredoxin showed a slow moving colourless material migrating behind the f a s t moving coloured native ferredoxin ( 3 3 1 . This was not observed with Sambucus ferredoxin ( F i -gure '7, Gel A). A p a r t i a l l y deteriorated preparation with a 4 2 3 / 2 7 7 nm absorbance r a t i o of only 0 . 2 6 8 s t i l l showed a single band. The appearance of an additional t r a i l i n g band i n 1 6 % polyacrylamide gels was reported for pure spinach ferredoxin ( 3 4 ) . This e f f e c t was observed with Sambucus f e r -redoxin during preliminary experiments with higher a c r y l -amide concentrations, but was not investigated further. In t h i s study ferredoxin was detected i n the a c r y l -amide gels by i t s native red brown colour and by the general protein s t a i n , Buffalo Blue Black (Amido Schwartz). In a polyacrylamide electrophoretie study of ferredoxin and other leaf proteins, Rouge ( 9 3 ) used the putatively more sensi t i v e s t a i n , Coomassie B r i l l i a n t Blue ( 9 4 ) . A recently reported s t a i n i s s p e c i f i c for iron-containing proteins. B r i l l et. al. have shown that ferredoxin, separated i n polyacrylamide gels and stained with 0.7% a,a-dipyridyl containing 8% mercapto-acetic acid, can be detected as a thin pink band ( 9 5 ) . Fluorescamine-labeled preparations of Sambucus f e r -redoxin, when fractionated i n SDS polyacrylamide gels by elec-trophoresis, showed only one fluorescent band. This was fur-ther convincing evidence of homogeneity since Fluorescamine I 4-phenylspiro(furan-2[3H],1 1-phthalan)-3,3'-dione] i s a highly sensitive reagent for the detection of peptides and proteins i n the picomole range ( 9 6 ) . Contaminating moieties with free 97 primary; aitii.no. groups were not present. P u r i f i e d Sambucus ferredoxin. was found to be s i m i l a r to other plant ferredoxins in. i t s spectral, properties, bio-l o g i c a l a c t i v i t y , amino acid composition, molecular weight, and active centre. The UV and v i s i b l e spectrum of native. Sambucus f e r -redoxin (Figure 5 , curve A) i s almost i d e n t i c a l to those of other plants (Table I I ) . The position of each absorption ma-ximum (peak) i s within 1 to 6 nm of the corresponding maxima of other ferredoxins.. The " c r i t i c a l r a t i o s " of. absorbances at these maximal positions, are also s i m i l a r to the r a t i o s re-ported for other angiosperm ferredoxins (Table III).. From extensive studies of the spectral properties of ferredoxin from a l f a l f a , Keresztes-Nagy and Margoliash con-cluded that the protein, the ir o n , and the l a b i l e sulphur account for the entire spectrum (33). No prosthetic groups such as f l a v i n e x i s t . The aromatic amino acid residues (Trp, Phe, Tyr) and the two iron atoms account for the entire ab-sorption at 277 nm. Angiosperm ferredoxins a l l contain seven aromatic residues (Table VIII, and r e f . 20). Their c r i t i c a l absorbance r a t i o s are sim i l a r to each other and.differ from those of fern (80) and algae (Table III) which lack trypto-phan and which have d i f f e r e n t t o t a l amounts of phenylalanine and tyrosine. The aromatic amino acid content of Sambucus f e r -redoxin was calculated to be only s i x residues: 1 Trp, 3 Tyr, 2 Phe (Table VII). The spectral s i m i l a r i t y of the protein to other angiosperm ferredoxins may indicate that i t instead con-tains seven aromatic amino acid residues. Tyrosine may 98 a c t u a l l y occur a§ four.. residue§..because its,. value of 3.48 residues, per molecule i n Table VII shows poor i n t e g r a l s t o i -chiometry. However, the content of phenylalanine i s also suspect and must await further confirmation because during the amino acid analyses i t showed the greatest difference i n recovery (20.4%) between the dried and undried protein hydro-lyzates (Table v i ) . Such anomalous recovery behaviour gives reason for further study. The true amino acid, composition of Sambueus ferredoxin ascertained from i t s complete sequence would indicate whether the UV absorption at 277 nm i s i n fact due to s i x or seven residues, together with the two atoms of iro n . The two l a b i l e sulphur atoms from the active centre of ferredoxin account for the structure of the near u l t r a v i o l e t and v i s i b l e portions of the spectrum (33). Deterioration of the protein i n ion-low water'involves the loss of the l a b i l e sulphur which re s u l t s i n the almost complete lack of absor-ption i n t h i s region. D e t e r i o x a t e & n S a m b u c u s ferredoxin (Figure 5, curve B) shows a spectrum not.unlike that of deter-iorated ferredoxin from Mediaago and Zea mays (33, 35). The molar extinction c o e f f i c i e n t at 423 nm varied de-pending on the method of c a l c u l a t i o n of the concentration term. Dry weight analysis indicated a range of 10.8 to 12.9 mM^cirf 1 for £ 1 * 2 3 . Iron analysis showed £ 4 2 3 as 9.1 mM 2cm 1 , a value similar to other plant ferredoxins (Table IV). Amino acid analysis of an aliquot of the solution being used for absorption measurement could give a more accurate estimate of the con-centration of the solution, but l i m i t e d amounts of the material 99 precluded t h i s . More extensive evidence regarding the extinc-t i o n at 423 nm should also involve an accurate determination of the l a b i l e sulphur content of Sambucus ferredoxin as thi s i s the source of the absorption i n t h i s region of the spectrum. Ferredoxins are defined as low molecular weight protein electron c a r r i e r s that transfer to appropriate enzyme systems some of the most "reducing" electrons i n c e l l u l a r metabolism (15). A useful test to i d e n t i f y ferredoxins i s th e i r a b i l i t y to catalyze the photoreduction of NADP.by washed chloroplasts. In t h i s respect, the protein p u r i f i e d from Sambucus leaves behaved i n a manner sim i l a r to other.plant ferredoxins (Figure 6). Spinach and corn ferredoxins both give i n i t i a l reduction rates of 100 umoles of NADP per mg chlorophyll per hr (35). Sambucus ferredoxin exhibited a somewhat.lower rate of 86 umoles NADP per mg chlorophyll per hr. This may have been caused by the ferredoxin sample being s l i g h t l y denatured. The absorbance r a t i o (423/277 nm) was only 0.44- (Figure 6). This represents a proportional. 0.12 "degree of deterioration" based on the maximal (0.49, Table III) and the minimal (0.075, Figure 5) ra t i o s obtained for the native and completely deteriorated protein respectively (33). Using t h i s as a correction value, the rate for native Sambucus ferredoxin. may be 98.2 umoles of NADP per mg chlorophyll per hour, very s i m i l a r indeed to that of corn and spinach. The amino acid compositions of a l l ferredoxins reported to date have been determined from analyses of desiccated or rotary evaporated hydrolyzates. This removal of excess acid p r i o r to analysis has been the usual procedure (97). In a re-cent study of th i s step using standard amino acid solutions, Robel has shown that the majority of amino acids incur s i g n i -f i c a n t losses through adsorption to the glass surface of the hydrolysis tubes (40). The errors were eliminated by omitting the drying step and d i l u t i n g the acid hydrolyzates d i r e c t l y with buffer. The acid hydrolyzates of Sambucus ferredoxin were treated both ways, as a preliminary investigation of the e f f e c t of drying on protein samples. The o v e r a l l r e s u l t was an i n - . crease i n the recovery of amino acids with undesiccated hydro-lyzates (Table VI). However, the r e l a t i v e average o v e r a l l amino acid increase of 7.3% i s d i f f i c u l t to re l a t e to Robel's f i n d -ings, as his average o v e r a l l amino acid loss due to evapora-t i o n (2.09%) was expressed i n r e l a t i o n to the absolute i n i t i a l amounts of standard, amino acids. Robel also found that asp-a r t i c acid, glutamic acid, glycine, and alanine were adsorbed more than other amino acids. With ferredoxin, aspartic acid, prol i n e , valine, and phenylalanine gave the greatest increases i n recovery. The reason for these differences i s unknown. Protein hydrolyzates may behave d i f f e r e n t l y from standard amino acid solutions. As the silicon-oxygen bridges of the glass surface are implicated i n the adsorption phenomenon, the com-posi t i o n of the Pyrex glass used for the hydrolysis tubes may have an e f f e c t . The reu l t s presented for Sambucus ferredoxin are only preliminary and should be considered tentative. The ef f e c t of drying on hydrolyzates may be better understood when studies with other proteins become ava i l a b l e . The amino acid compositions of ferredoxins (e.g. Medi-101 eago (33) , Soenedesmus (45) , Leuoaena (72) , Cypevus (75)) have been based on average or extrapolated data from hydrolyzates of d i f f e r e n t times. Extrapolation to zero time corrects for the usual decomposition of l a b i l e amino acids such as serine, threonine, cysteine, and tyrosine (97) . Valine, leucine, and isoleucine, with hydrophobic side chains,.form peptide bonds less susceptible to hydrolysis than other amino acids and are released i n increasing amounts with time. The res u l t s with Sambueus ferredoxin showed that most, of the amino acids, i n c l u -ding the l a b i l e ones, were recovered i n maximal amounts afte r the longest period of hydrolysis (Tables V, VI). The presence during hydrolysis of the iron and sulphur from the active centre of the protein may have caused t h i s unusual pattern of recovery. The maximal values were- flsedtfor the cal c u l a t i o n s . The amino acid composition presented i n thesresults was based on the combined data from both dried and undried hydrolyzates because the limited number of r e p l i c a t e s i d i d r n o t allow a rigorous assessment of either method. Also for com-parative purposes, i t was f e l t necessary to base the amino acid composition of Sambueus ferredoxin. p a r t l y on the analyses of dried hydrolyzates because a l l previous reports of ferredoxin composition have involved the analyses of dried hydrolyzates. The amino acid composition was calculated using an average value (70.13 nmoles amino acid per mg protein)"from those amino acid residues whose i n t e g r a l stoichiometry most clo s e l y resembled that of the residue occurring i n the smallest amount (arginine). The compositions of other ferredoxins have been calculated i n a sim i l a r manner (33, 35, 75). Determination 102 of the best integer r a t i o s of amino acid residues can now be f a c i l i t a t e d by a computer method published recently (98) Sambucus ferredoxin has an amino acid composition nearly i d e n t i c a l to those of other plants (Table VIII, and r e f . 20), which i n general show c h a r a c t e r i s t i c s s i m i l a r to those found for the b a c t e r i a l ferredoxins (13). In alIdeases•the a c i d i c and a l i p h a t i c amino acids are preponderant, i n contrast to the low content of basic and aromatic residues . Tryptophan i s missing i n a l l ferredoxins from non-angiosperm plants. The occurrence of methionine i s sporadic. Thus far only one residue of methionine per molecule has been reported for Cyperus3 Ama-ranthus3 and Gossypium of the angiosperms, and Microcystis3 Bumelleriopsis3 Scenedesmus3 and Equisetum of the lower plants. The ferredoxins of Cladophora' and Polystichum contain two re-sidues of methionine (20). Methionine i s absent from Sambucus ferredoxin as i t i s from f i v e other angiosperms (Table VIII). An important aspect of the amino acid composition i s the pre-sence of only four cysteine residues,.which are s u f f i c i e n t to form the iron-sulphur c l u s t e r of the proposed form (99). Gossypium3 Cyperus, and Zea mays also contain ferredoxins with only four cysteine residues. The systematic comparison of amino acid compositions of ancestrally related proteins i s greatly aided by the use of multivariate analysis methods. One such method which i s simple to use i s that of Metzger et. al. (100). A "difference index" inversely proportional to the compositional relatedness between two proteins i s calculated by obtaining the difference i n the molar f r a c t i o n a l contents of each amino acid, summing the 103 absolute values of those differences, and multiplying that sum by f i f t y . Two proteins with no amino acid i n common have a difference index (DI) of 100. Two proteins with the i d e n t i c a l composition have a DI of zero. This method has been used ex-tensively during recent years i n comparisons of a wide variety of proteins [e.g. 30S ribosomal proteins (101), dehydrogenases (102), Brassiea seed globulins (103)]. Both.Slobin (101) and Fondy and Holohan (102) have used amino acid sequence data from cytochromes e to show that compositional DI up to a l e v e l of approximately 10 correlates very c l o s e l y with sequence s i m i l a r -i t y . Not enough sequences of ferredoxins.are available to thor-oughly test the method with t h i s group of proteins. However, a preliminary analysis of the amino acid compositions of Sam-bucus ferredoxin and sixteen other plant ferredoxins (data from ref . 20) showed that Sambueus ferredoxin i s most clos e l y related to that of L eueaena (Rosales, DI — 7 .797) , then Colocassia (Ar— ales) and • Medieago • (Rosales, both with DI = 8 .247), and then Zea mays (Graminales, 9.484). The composition most d i s t a n t l y related to Sambueus ferredoxin i s that of Anaoystis (blue-green alga, DI = 18.046). The remaining ten ferredoxins compared to Sambueus give DI values ranging from 10.429 to 17.235. The f i v e plant ferredoxins for which the complete primary structure i s known possess sequence differences ranging from 16% to 35% (1.9). The correlation, between t h e i r difference indices and t h e i r percent sequence differences might be used to "predict" the s t r u c t u r a l r e l a t i o n s h i p of Sambueus ferredoxin to those ferredoxins already sequenced. The successive comp-arison of a l l f i v e sequenced ferredoxins shows that Sambueus 1 ferredoxin may exhibit a 16, 17, 18, 25, and 38% sequence d i f -ference with Colocassia, Leucaena, Medicago, Spinacea (Cheno-podiales), and Soenedesmus .(green alga) respectively. I t must be stressed that these results are highly speculative and only are presented here to i l l u s t r a t e the remarkable simi-l a r i t y of the amino acid composition of Sambucus ferredoxin to those of other angiosperm ferredoxins. .The l i m i t a t i o n s and values of Metzger's DI method have.been discussed (102). However, the compositional, relatedness of ferredoxins deserve further study, especially i n view of the d i f f i c u l t y of ob-taining sequence data, and the use of other multivariate tech-niques should also be investigated with t h i s class of proteins (104, 105, 106). The molecular weight of Sambucus ferredoxin (10,74 0) i s s i m i l a r to those of other plant ferredoxins (20) and almost i d e n t i c a l . t o the formula molecular weights of those plant ferredoxins which have been sequenced (19). U l t r a c e n t r i f u -gation, Sephadex G-75 gel f i l t r a t i o n , and determination of iron content have been commonly used, methods to estimate the molecular weights of ferredoxins (35, 73, 20). Besides being calculated from the amino acid composition, the molecular weight o f . S a m b u c u s ferredoxin was determined by.the recently developed technique of SDS electrophoresis of fluorescent' proteins. The excellent agreement between the values derived from these two sources indicates the s e n s i t i v i t y of the l a t t e r method. The l i n e a r r e l a t i o n s h i p between the logarithm of the molecular weights of the standards and t h e i r electrophoretic mobility (Figure 8) was taken as adequate evidence of the r e l i 105 a b i l i t y of the method. However, i t was recognized that the migration of an additional standard such as parathyroid hor-mone (molecular weight 9,000) ahead of the unknown samples could 'have lent a further degree of accuracy to the estimation of the molecular weight of Sambueus ferredoxin. The iron content presented i n the r e s u l t s was based on analyses of ferredoxin samples whose concentration was de-termined, by dry weight (Table IX). Others have used amino acid analysis of an aliquot of the sample or the molar extinction c o e f f i c i e n t to determine the ferredoxin concentration used i n the c a l c u l a t i o n of the iron content (45, 80, 81). The values obtained for Sambueus (2 g atoms of iron per molecule) are i n good agreement with those for other plants (5). To obtain further evidence.of the t y p i c a l nature of the active centre i n Sambueus ferredoxin, the content of l a b i l e sulphur must be accurately determined. Also, electron paramagnetic (EPR) and electron nuclear double resonance (ENDOR) spectroscopy of Sambueus ferredoxin would y i e l d invaluable.information con-cerning physico-chemical properties of i t s . a c t i v e centre (107). The sequence data presented i n the results also show that Sambueus ferredoxin i s very s i m i l a r to other angiosperm ferredoxins. The amino-terminal residue i s alanine. A l l higher plant ferredoxins investigated have the same amino-terminal residue except Cypevus i n which i t i s serine (19, 74, 75). The amino-terminal sequence of Sambueus ferredoxin i s Ala-Thr. The same sequence has only been reported for Colooassia and Seenedesmus (19). Thermolytic peptide Th-1, Leu-Asx(Asx, Glx, Glx) , showed a net. charge of +1 at pH 1.9.. This peptide appears to be homologous to the peptide chain of Colocassia and Leucaena at position 64-68 where the sequence i s Leu-Asp-Asp-Glu-Gln (see Appendix). The placement of t h i s peptide i s consistent with the cleavage s p e c i f i c i t y of thermolysin (50) because residue 69 i s isoleucine i n a l l four.sequenced angio-sperm ferredoxins. The sequence at pos i t i o n 64-68 i n both Spinacea and. Me die ago i s almost i d e n t i c a l , Leu-Asp-Asp-Asp-Gln (19). The amino-terminal sequence of peptides Th-2 and Th-6 was Leu-Gly. Position 31-32 i n Leucaena i s the only oc-currence of this.sequence i n the f i v e ferredoxins which have been sequenced. The composition of peptide Th-3 suggested that itatwas. was tri p e p t i d e so the complete sequence must be Leu-Thr-Ala. This sequence or a s i m i l a r i l y substituted one only occurs i n ferredoxins at position .9.5-97 where the actual sequence i s Leu-Thr-Ala i n a l l four angiosperms. This strongly suggests that peptide Th-3 represents the carboxyl-terminal sequence of Sambucus ferredoxin. Homology with other ferredo-xins allows the assignment of peptide Th-4, Ile-Asx (Glx, Gly, Trp, Val) to position 69-74. The sequence i n Spinaeea i s Ile-Asp-Glu-Gly-Trp-Val. The placement of peptide Th-4 i s also consistent with thermolytic s p e c i f i c i t y as residue 75 in a l l sequenced ferredoxins i s leucine. The Tyr-Leu sequence at the amino-terminuS' of peptide Th-5 i s unique to Sambucus ferredoxin. Although the above tentative sequence r e l a t i o n -ships considered together, indicate a close s i m i l a r i t y between Sambucus ferredoxin and other plant ferredoxins, i t i s clear that the ferredoxin.sequence from Sambucus. cannot be i d e n t i c a l to any of the known sequences. Only the elucidation of the 107 complete primary structure can reveal the degree of divergence of Sambueus ferredoxin from those of other plants. To t h i s end, the sequencing of peptides from thermolytic digests of TCA-treated ferredoxin appears to be a promising approach.. Rao and Matsubara obtained 25 thermolytic peptides i n good y i e l d from performic acid-oxidized ferredoxin (49). The strategies used for sequencing other ferredoxins have had much i n common (49, 50, 72, 86, 87). Generally, t r y -p t i c and chymotryptic peptides from TCA-treated or carboxy-methylcysteinyl (CMCys) ferredoxin were separated by ion-exchange chromatography on columns of Dowex AG 1-X2 and p u r i f i e d by high voltage paper electrophoresis. Due to the low content of basic and aromatic residues i n the protein, most of these peptides were large and required further cleavage with additional en-zymes. But conversion of the four or f i v e cysteine residues i n ferredoxin to AECys makes these s i t e s also available for t r y p t i c cleavage (47). Matsubara and Sasaki characterized 17 t r y p t i c peptides from spinach AECys-ferredoxin (50). I t was thought that a similar digest of Sambueus AECys-ferredoxin would y i e l d an adequate number of small peptides e a s i l y se-parated by gel f i l t r a t i o n and paper electrophoresis, thus obviating the d i f f i c u l t i e s of ion-exchange chromatography with v o l a t i l e buffers. However, the use of AECys-ferredoxin was not f r u i t f u l as t h i s derivative was insoluble. These re s u l t s suggest that the use of performic acid-oxidized and carboxymethylcysteinyl (CMCys) derivatives may prove more useful i n theas.equencing of Sambueus ferredoxin. 108 The separation of t r y p t i c and chymotryptic peptides from these sources may require ion-exchange chromatography. The advant-age of following a strategy similar to that used with other ferredoxins i s the increased ease with which nonhomologous peptides can be i d e n t i f i e d and sequenced. Ferredoxin from Sambucus racemosa (Caprifoliaceae) resembles other plant ferredoxins i n i t s spectral properties, molecular weight, iron content, and electron-transfer a c t i -v i t y . The amino acid composition, t y p i c a l of t h i s group of proteins, i s highly s i m i l a r to those of ferredoxins from other angiosperms, and most c l o s e l y related to that of Leu-caena (Leguminosae). The amino-terminal amino acid sequence i s i d e n t i c a l to that of Colocassia and- S c e n e d e s m u s. The carboxyl-terminal amino acid sequence i s probably i d e n t i c a l to that of Leucaena, Colocassia}3 Spinacea, and Medicago. P a r t i a l i n t e r n a l amino acid sequences are also c h a r a c t e r i s t i c of angiosperm ferredoxins. Elucidation of the complete amino acid sequence w i l l determine the actual degree of s i m i l a r i t y between Sambucus ferredoxin and other plant f e r -redoxins. The i s o l a t i o n and p u r i f i c a t i o n methods developed i n the course of the present E e s e a r c h w i l l allow the prepara-t i o n of Sambucus ferredoxin i n quantities large enough to f a c i l i t a t e the further characterization of the molecule and the determination of i t s complete amino acid sequence. APPENDIX THE AMINO ACID SEQUENCE OF FERREDOXIN FROM THREE HIGHER PLANTS Spinaoea 1 Ala 2 ALA 3 TYR 4 Lys 5 Val 6 THR 7 Leu 8 VAL 9 Thr 0 Pro 1 THR 2 3 Gly ASN 4 VAL 5 Glu 6 Phe 7 GLN 8 Cys 9 Pro 0 Asp' Colooassia Ala THR TYR Lys Val LYS Leu VAL Thr Pro SER Gly GLN GLN Glu Phe GLN Cys Pro Asp' Leuoaena - ALA PHE Lys Val LYS Leu LEU Thr Pro ASP Gly PRO LYS Glu Phe GLU Cys Pro Asp' 4 0 2 1 2 3 4 5 6 7 8 9 3 0 1 2 3 4 5 6 7 8 9 Sp. Asp Val Tyr H e Leu Asp ALA Ala Glu Glu GLU Gly H e ASP Leu Pro Tyr Ser Cys Arg' . . Co. Asp Val Tyr H e Leu Asp GLN Ala Glu Glu VAL Gly H e ASP Leu Pro Tyr Ser Cys Arg Le. Asp Val Tyr H e Leu Asp GLN Ala Glu Glu LEU Gly H e GLU Leu Pro Tyr Ser Cys Arg' Sp. 4 1 Ala 2 Gly 3 Ser 4 Cys 5 Ser 6 Ser 7 Cys 8 Ala 9 Gly 5 0 Lys 1 LEU 2 LYS 3 THR 4 Gly 5 SER 6 LEU 7 ASN 8 Gin 9 ASP 6 0 Asp Co. Ala Gly Ser Cys Ser Ser Cys Ala Gly Lys VAL LYS VAL Gly ASP VAL ASP Gin SER Asp , Le. ALA GLY SER CYS SER SER CYS ALA GLY LYS LEU VAL GLU Gly ASP LEU ASP Gin SER Asp Sp. 1 GLN 2 Ser 3 Phe 4 Leu 5 Asp 6 Asp 7 ASP 8 Gin 9 H e 0 ASP 1 Glu 2 3 Gly Trp 4 Val 5 Leu 6 Thr 7 Cys 8 ALA 9 Ala 0 Tyr Co. GLY Ser Phe Leu Asp Asp GLU Gin H e GLY Glu Gly Trp Val Leu Thr Cys VAL Ala Tyr Le. GLN Ser Phe Leu Asp Asp GLU Gin H e GLU Glu Gly Trp Val Leu Thr Cys 9 7 Ala ALA' Ala Tyr Sp. 8 1 Pro 2 VAL 3 Ser 4 Asp 5 VAL 6 THR 7 H e 8 Glu 9 Thr 9 0 His 1 Lys 2 Glu 3 Glu 4 Glu 5 Leu 6 Thr Co. 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