New Phytl. (1967) 66, 47-56. FINGERPRINT PATTERNS F THE GLBULIN FRACTIN BTAINED FRM SEEDS F VARIUS SPECIES F THE FABACEAE BY P. JACKSN, J. M. MILTN AND D. BULTER* The Hartley Btanical Labratries, University f Liverpl {Received <) July 1966) SUMMARY The patterns (fingerprints) f the peptides separated frm tryptic digests f the glbulin fractin f seeds ffivegenera f the \'icieae have been cmpared and shwn t be very similar. Patterns btained with Phaselus vulgaris, and tw species f Canavalia (Phaseleae, seiisu lat), were different frm thse f the Vicieae; the tw Canavalia species gave an identical pattern. Fingerprints f the glbulin fractin f Abrus precatrius (Abreae) differed frm thse f Phaselus vulgaris and als frm thse f the varius members f the Vicieae. The usefulness f this methd fr supplying data fr systematic purpses is discussed. INTRDUCTIN As lng ag as 194, Nuttall pinted ut the pssible usefulness in systematics f a cmparisn f the structures f hmlgus prteins extracted frm difterent rganisms. Within the last decade r s such studies have becme increasingly cmmn (see, fr instance, varius articles in Lene, 1964, and Brysn and Vgel, 1965). There are seven main methds presently used t cmpare prtein structures: (i) amin acid cmpsitin, (2) fingerprint pattern f peptides, (3) amin acid sequence, (4) catalytic activity, (5) immunlgy, (6) electrphresis, (7) chrmatgraphy. Each methd has varius advantages and disadvantages frm the standpint f their usefulness in systematics (Sibley, 1962; Wilsn and Kaplan, 1964) and when pssible a cmbinatin f methds shuld be emplyed. The fingerprinting technique used t btain the results given in this paper has been chsen since n the ne hand it yields many mre 'bits' f infrmatin fr cmparisns than d the quicker electrphretic methds whilst n the ther band it is nt nearly s time-cnsuming as the mre infrmative determinatin f amin acid sequence. Fingerprinting was first used by Ingram (1958) t shw a single amin acid difference between tw haemglbin mlecules. Mre recently Zuckerkandl, Jnes and Pauling (i96) cmpared the structures f the haemglbin f primates, fishes and invertebrates by fingerprinting. Later wrk by Zuckerkandl and Schreder (1961) and Rifkin and Knigsberg (1965) n the amin acid sequence f haemglbin frm these rganisms cnfirmed Zuckerkandl's earlier cnclusins abut the relatinships between these animals based n fingerprint data. Wilsn and Dixn {1961) fingerprinted insulin mlecules frm difterent telest fish and shwed that they differed radically frm mammalian insulins. Hwever, the wrk f Kendrew et al. (1964) and Stckwell (1961) n myglbin and f Anfinsen et al. (1959) n ribnuclease shw that fingerprint data can smetimes be * Present address: Department f Btany, Suth Rad, University f Durham. D NP 47
48 P. JACKSN, J. M. MILTN AND D. BULTER misleading in systematic studies. With bth these prteins the results they btamed with this methd were nt in accrd with serlgical, anatmical and palaentlgical findings. It is likely, therefre, that fr myglbin and ribnuclease fingerprinting cmparisns are a relatively pr guide t similarity r therwise f prtein structure. MATERIALS AND METHDS Alaterials Chemicals were btained frm British Drug Huses Ltd, Ple, Drset, and were f analytical grade except Trypsin (3 x recrystallized, salt free, sterile) which was frm Wrthingtn Bichemical Crpratin, Freehld, New Jersey, and A^-ethyl mrphline frm Kdak Ltd, Kirkby Trading Estate, Liverpl. -5 1 15 2 3 Time (hurs) Fig. I. Time curse fr the enzymic hydrlysis f the carbxymethylated glbulin fractin f Vicia faba seeds. The degree f hydrlysis was fllwed by determining the amunt f TCA sluble material using the methd f Itzhaki and Gill (1964). (Each value is the average f three determinatins.) The vertical axis, E3i ^m,, is the ptical density' f the slutin at 31 m/i in a cell f i cm light path. The bilgical material was btained frm the fllwing surces: Vicia faba, Pisum sativum and Phaselus vulgaris irm Friz C. Ltd, Birkenhead; Abritsprecatarius irm Thmpsn and Mrgan Ltd, Ipswich; Vicia dumetrum, V. angustiflia and V. sativa frm The Ryal Btanic Gardens, Kew; Lathyrus clymenum, L. sativus, and L. sylvestris frm Wye Cllege, Lndn University; Canavalia ensifrmis frm British Drug Huses Ltd, Ple, Drset; C. gladiata frm Department f Btany, University f Ghana. N attempt was made t check the identity f these species. Methds Ctyledns f saked seeds were separated frm the testa and embry axis and grund with acid-washed sand in a mrtar. The glbulin fractin was extracted frm the paste and partially purified by the methd f Danielsn (1949). The dialysed salt-free
Glbulin fractin f Fabaceae 49 glbulin was carbxymethylated by the methds f Sela, White and Anfinsn (1959) and Harris (1964). Fifty mg f the carbxymethylated glbuhn was disslved in t; ml.34 M A'^-ethyl mrphline (NEM) buffer, ph 7.9, made 8 M with respect t urea. This slutin was made t 5 ml with buffer, i mg f trypsin added and the mixture incubated fr 2\ hurs at 37 C. This ptimum time fr digestin, 2-1- hurs, was determined by measuring the increase, with time, in ptical density f the TCA sluble material using the micrbiuret methd f Itzhaki and Gill (1964) (see Fig. i). The digestin mixture was lyphilized, redisslved in a minimum vlume.1 M NH3 and fractinated n a Sephadex G-25 (bead) clumn (135 x.9 cm) using.i M NH3 as eluant. Fractins (3.5 ml) were cllected and their absrptin at 28 m/( measured in silica cells f i cm light path using a Unicam Sp.5 spectrphtmeter (see Fig. 2). Fractins 8-14 were cmbined and fingerprinted using the methd f 1-8 2 15 12" U 16 18 2 Tube number Fig. 2. Elutin diagram f peptide mi.xture after separatin n Sephadex G-25. The vertical axis, Ejg'mfi '^ '^he ptical density f the slutin at 28 m/; in a cell f i cm light path. Margliash and Smith(i962). Chrmatgrams were dipped in cadmium acetate ninhydrin reagent (Atfield and Mrris, 1961) and left vernight t detect the psitin f peptides. Initial experiments have shwn that fractins 1-8 cntained twelve acidic, tgether with a few basic, peptides. Mst f the tryptphane and tyrsine residues cntained in the glbulin fractin ccurred in these peptides; but due t their large size these peptides gave large diffuse spts s that reprducible fingerprints were difficult t btain. Fractins 14-18 cntained mainly urea and a few amin acids. Fractins 8-14 cntained the neutral and basic peptides apart frm the few btained in fractins 1-8. It is fr these reasns that nly fingerprints f fractins 8-14 are present in Figs. 3 and 4. Presentatin f residts Peptide maps were traced nt thin paper and the tracings phtgraphed and reduced in size. The patterns btained were cmplex and in rder t facilitate interpretatin they were each divided int six sectins whse limits were drawn thrugh areas f lw spt density. When cmparing tw fingerprints the equivalent sectins in
5 P. JACKSN, J. M. MILTN AND D. BULTER each fingerprint were cmpared with each ther s as t achieve a satisfactry general cmparisn. If peptide digests frm tw different glbulins gave similar patterns n fingerprinting, then the digests f each were mixed in equal prprtins and fingerprinted tgether. The pattern btained fr the mixture was then cmpared with the patterns btained fr the tw individual cmpnents. Spts cmmn t all members f the Vicieae are blacked in n the figures except in Fig. 3(a) and (b). '^i (a) C ~b ^^^" ^ r)ap ^f Q c? Q^ c^ r (b) (c) (d) (e) Fig. 3. Fingerprint patterns f varius species f the Vicieae. (a) Vicia faba cv. Sharps Cnquerer; (b) V. faba cv. Masterpiece Green Lngpd; (c) V. dunietrum; (d) Lathvrus sylvestris; (e) Vicia angustfua and Lathy nis sylvestris; (i)lens esculent a. Sample sptted in the tp left crner, electrphresis frm left t right and chrmatgraphy in the ther directin. Y = yellw.
Glbulin fractin f Fabaceae c \ RESULTS Reprducibility f the technique When ahquts f the same enzymic digest f Vicia faba glbulin are fingerprinted there is sme variatin in the patterns btained. This variatin always ccurs at the edge f sectins II and III nearest t sectin I wbere there are several weak diffuse spts which are nly smetimes delineated; the number f these spts is never greater than five. Similarly when the glbulin fractin extracted frm different samples f the same batch f beans are fingerprinted the variatin encuntered is f the satiie rder as that already described and invlves peptides in the same area. Table i. The number and distributin f the peptides in fingerprints f the glbulin fractin f varius members f the genus Vicia Sectin I II III IV VI N. f different spts cmmn t all I'icia spp. 7 pink, I yellw 6 pink 12 pink, I yellw 7 pink, I yellw 13 pink, 2 yellw 6 pink N. f spts nt present in all fur spp. Species in which the spts nt present in all fur species are fund (i) V.faba, F. dumetruin (i) V. angustiflia (1) V.faba, I (2) V. faba, I (3) V.faba, I (4) V.faba, I (5) V.faba (6) (7) (8) V. sativa sativa V. dunietruni sativa, V. angustiflia sativa, V. angustiflia sativa angustiflia (9) V. angustiflia (1) V.faba, V. dumetruni (2) V. faba, V. anqustiflia (3) V.faba (1) V. faba, V. angustiflia (2) V. faba, V. angustiflia (3) V.faba, V. dumetruin (4) V. angustiflia, V. sativa (5) V. angustiflia (1) V.faba (2) V. angustiflia The numbers in parentheses in the last clumn identify a particular peptide, i.e. Sectin III (i) this peptide is cmmn t V. faba, V. sativa and V. angustiflia. Reprducibility f the bilgical material When the glbulin fractin frm tw samples btained frm different hara'ests f a cultivar were fingerprinted the patterns btained were identical within the Hmits mentined abve. Tbis was als fund t be the case when five different cultivars f V. faba were subjected t fingerprint analysis. The maps fr tw f these cultivars, V. faba cv. Sharps Cnquerer and V. faba cv. Masterpiece Green Lngpd are given in Fig. 3(a) and (b). Cmparisn f the fingerprints frm different genera f the Fabaceae Vicieae. Fig. 3(c) gives the fingerprint pattern f V. dumetrum and the results in Table i are a detailed analysis f the spt data btained when fur species f Vicia were cmpared. The results in this table shw that seventy-seven different spts were btained f which fifty-six were cmmn t all f the fur species, eleven were present in mre than
52 p. JACKSN, J. M. MILTN AND D. BULTER ne but nt in all f the species and ten were present in ne species nly. f the ten spts which were fund in nly a single species, six ccurred m the area f sectin II and III mentined befre. r T- ui Fig 3(d) gives the fingerprint pattern f Lathyrus sylvestris and the results t 1 able 2 are a detailed analysis f the spt data btained when fur species f Lathyrus were cmpared. The results shw that eighty-tw different spts were btained, fifty-seven f which were cmmn t all f the fur species, fifteen were present in mre than ne but nt in all f tbe species and ten were present in ne species nly. f the ten spts which were fund in nly a single species six ccurred in the area f sectin II and III mentined befre. Table 2. The mtmber and distributin f the peptides in fingerprints f the glbulin fractin f varitts species f the genus Lathyrus Sectin I II in IV V VI N. f spts cmmn t all Lathyrus spp. 7 pink, I yellw 5 pink 13 pink, I yellw 7 pink, I yellw 14 pink, 2 yellw 6 pink N. f spts nt present in all fur spp. I 3 12 3 4 2 Species in which the spts nt present in all fur species are iund (i) (I) (2) (3) (I) (2) (3) (4) (5) (6) (7) (8) (9) (1) (11) (12) (i) (2) (3) (i) (2) (3) (4) (i) (2) L. syivestris. L. syivestris, L L. sylvestris, L. syhestiis. L. apiiaca L. sytvestris. L. sytvestiis. L. syh'estris L. ciymentim L. sativa, L. L. sylvestris L. clymenum L. sativiis L. sativa, L. L. sativa, L. L. sativa, L.. sylvestris L. sativa L. ciymenum L. clymenum L. clymenwn L. sativa, L., L. clvmenum ciymenum ciymenum clymenum aphaca, L. sativa, L. sylvestris aphaca The numbers in parentheses in the last clumn identify a particular peptide, i.e. sectin IV (i) this peptide is cmmn t L. sylvestris, L. sativa and. A mixture f the digests f Vicia angustiflia and Lathyrits sylvestris (Fig. 3e); Vicia dumetrum and Lathyrus clymenum; and Vicia faba and Lathyrus aphaca were fingerprinted and fifty-fur f the fifty-seven spts cmmn t all genus Lathyrus were in the same psitins as fifty-fur f the fifty-six spts cmmn t the genus Vicia. The fact that the patterns f species frm these tw genera virtually cincide when they are fingerprinted tgether indicates that the fingerprint patterns f these tw genera are directly cmparable. The glbulin fractin f Lens esculenta (Fig. 3f) when fingerprinted gave a pattern in which fifty-three spts were in the same psitin as fifty-three f the fifty-six spts cmmn t the genus Vicia. Similarly Fisum sativum (Fig. 4a) and Cicer arietinum
Glbulin fractin f Fabaceae D (c) c ( ^ ^ Qi (d) (e) c? CXD Fig. 4. Fingerprint patterns f varius species f the Fabaceae. (a) Pisum sativum; (b) Cicer arietinum; (c) Canavaliaensifrmis; (d) C.gladiata; (e) Phaselusvulgaris; {i) Abusprecatrius. Sample sptted in the tp left crner, electrphresis frm left t right and chrmatgraphy in the ther directin. (Fig. 4b) glbulins when fingerprinted had frty-seven and frty-ne spts respectively in the same psitin as the crrespnding nes f the fifty-seven spts cmmn t the genus Vicia. Phaseleae (sensu lat). The glbulins f tw species f Canavalia (tr, Phaseleae, (f)
54 P. JACKSN, J. M. MILTN AND D. BULTER sensu lat), C. en.^ifrmis (Fig. 4c) and C. gladiata (Fig. 4!) and ne species f Phaselus (tr, Phaseleae sensu lat), P. vulgaris (Fig. 4e) were als fingerprinted. The patterns btained differed frm thse f the Vicieae in that fewer spts (thirty-seven) were present in every case. The tw Canavalia species gave identical patterns which differed frm that f P. vulgaris. Abreae. The glbulin fractin f Abrus precatrius (placed by Hutchinsn, 1964, in the tribe Abreae) n fingerprinting resulted in a pattern with frty-eight spts (Fig. 4f) and the pattern differed cnsiderably frm that f Phaselus vulgaris and the varius genera f the Vieieae. DISCUSSIN When the structures f a single r f a few hmlgus prteins are cmpared fr systematic purpses, the expressin f nly a small part f the genetic infrmatin f the rganisms cncerned is being cnsidered. This, cupled with the fact that the techniques invlved are relatively time cnsuming, means that the particular chice f prtein investigated may prve t be critical. Earlier investigatins f this kind were mainly cncerned with nn-enzymic prtein, althugh varius authrs have pinted ut that enzymes are better suited fr systematic studies (Wilsn and Kaplan, 1964) than ther prteins. Hwever, there are tw majr disadvantages invlved in the use f enzymes. ne is the technical difficulty in btaining purified enzymes in sufficient amunts fr investigatin. The ther is that all previus mutatinal events in the evlutin f a species will nt be recrded in an essential mlecule, in which mst f the pssible amin acid substitutins wuld have given an inactive mlecule and hence led t the nn-survival f the individual cncerned. Thus the variatin encuntered in hmlgus enzyme mlecules might be crrespndingly less than the variatin encuntered in nn-enzymic prteins and cnsequently might be less readily detected. In many members f the Fabaceae, apprximately 2^ f the seed weight is due t the relatively well-defined and easily extracted glbulin fractin which cnsists essentially f strage prtein withut enzymic functin. It is fr the abve reasns therefre that this fractin has been used in the studies reprted here. Zuckerkandl and Pauling (1965) have suggested that glutenin, a majr seed prtein frm wheat, is nt a tertiary semantide. Hwever, the reprducible fingerprint patterns btained with the glbulin fractin here shw that the prteins f this fractin frm the varius species f the Fabaceae examined are definite entities; therefre it is reasnable t suppse that they are frmed by the nrmal template mechanism. W'hen aliquts f the same digest were fingerprinted the pattern was cnstant althugh the actual psitin f the peptide spts varied smewhat. In making cmparisns between fingerprints, therefre, it is the verall pattern whieh has been cmpared and nt the R( values f individual spts. The average psitin t which a peptide migrates is mainly dependent upn its charge and its partitin cefficient in the chrmatgraphic slvent mixture s that it is pssible fr different peptides t ccupy the same psitin n a peptide map. Hwever, when cmparing the fingerprints f clsely related species which give a very similar pattern, it wuld seem likely that the crrespnding spts in the fingerprints are the same r at least very similar peptides. We have fund that five genera f the Vicieae have a very similar pattern. Thse f Lathyrus and Lens differ frm Vicia each by three spts while thse f Pisum and Cicer differ frm Vicia by seven and thirteen spts respectively. The suggested relatinship
Glbitlin fractin f Fabaceae f Vicia, Lathyrus and Lens n the basis f spt data is in agreement with the views f many taxnmists. When fingerprint patterns f less clsely related taxa are cnsidered, ne cannt be s certain that crrespnding peptide spts are due t the same r similar peptides; ne can nly say whether r nt the patterns are different. Thus frm the results reprted here it is quite clear that patterns f the tw genera which were examined f the Phaseleae {sensu lat) were very different frm thse btained with genera f the Vicieae. It is f interest that the number f spts in the fingerprints f bth genera f the Phaseleae was the same, pssibly indicating that the psitins f arginine and lysine in the parent prtein fractin was the same fr bth genera. Klz and Turkva (1963) have shwn by an immunelectrphretic analysis that five genera f the Vicieae cntain prteins that are similar t r identical with Legumin and Vicilin f Pisum sativum, whereas bth types f prtein are absent in Phaselus vulgaris. The data reprted here cmplement and supprt the cnclusin that the glbulin fractins f the varius genera f the Vicieae are very similar and differ cnsiderably frm thse f P. vulgaris. A similar cnclusin has already been reached frm an electrphretic study f the glbulin fractins f these same rganisms (Bulter, Thurman and Derbyshire, 1967). If the crrelatin between the results f disc electrphresis and fingerprinting is generally applicable the ideal apprach might be t make a survey with the frmer technique and then apply the mre refined fingerprint methd in the light f the results btained. It is interesting t cmpare these results with thse f a DNA hybridizatin study n Pisum sativum, Vicia vilisa and Phaselus vulgaris which shwed that abut half f the nucletide sequences f the DNA f Vicia vilisa are similar t thse in Pisum whereas nly ne-fifth f the sequences f the DNA f Pisum and Phaselus are similar (Bltn et al., 1965). The spt data based upn glbulin prteins agree with the results f the DNA hybridizatin study since the fingerprints f Pisum and Vicia are mre similar than thse f Pisum and Phaselus. It is f nte that the fingerprint pattern f Abrus precatrius, althugh different frm that f members f the Vicieae, Phaselus vulgaris and Canavaiia spp., shws a greater similarity t the frmer rather than the latter. This is further supprt fr the cnclusin suggested by Bulter et al. (1967) n the basis f the electrphretic prperties f these glbulin fractins, that Abrus precatrius is related t but nt a member f the Vicieae. 5^ ACKNWLEDGMENT We shuld like t thank Prfessr V. H. Heywd fr several helpful cmments. REFERENCES ANFINSN, C. B., AQUIST, S. F. G., CKE, J. P. & JNSSN, B. (1959). A cmparative study f the structures f bvine pancreatic ribnucleases. J. bil. Chem., 234, 1118. ATFIELD, G. N. & MRRIS. C. J.. R. (1961). Paper electrphresis f amin acids. Bichem. J., 81, 66. BLTN, E. T., BRITTEN, R. J., CWIE, D. B., RBERTS, R. B., SZAERANSKI, P. & WARING, M. J. (1965). Yb. Carnegie Instn Wash., 64 (1964-6S), 313. BULTER, D., THURMAN, D. A. & DERBYSHIRE, E, (1967). A disc electrphretic study f glbulin prteins f legume seeds with reference t their systematics. Nei Phytl., 66, 27. BRYSN, V. & VGEL, J. J. (Eds.) (1965). Evlving Genes and Prteins. New Yrk. D.ANIELSN, C. E. (1949). Seed glbulins f the Gramineae and Leguminsae. Bichem. J., 44, 387. HARRIS, I. (1964). Structure and catalytic activity f alchl dehydrgenases. Nature, Lnd., 23, 3. INGR-WW, V. (1958). Abnrmal human haemglbins. Bichim. biphys. Acta, 28, 539. ITZHAKI, R. F. & GILL, D. M. (1964). A micrbiuret methd fr estimating prtein. Analyt. Bichem., 9, 41.
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