Reaction of Fluorescein-Isothiocyanate with Proteins and Amino Acids

Transcription

1 The Jurnal f BichemiBtry, Vl. 66, N. 6, 1969 Reactin f Flurescein-Isthicyanate with Prteins and Amin Acids II. Preparatin f FIurescein-Thihydantin Amin Acids and Their Thin-layer Chrmatgraphy By HIROSHI KAWAUCHI*, KATURA TUZIMURA*, HIROSHI MAEDA-f' ** and NAKAO ISHIDA** (Frm the Department f Fd Chemistry, Labratry f Analytical Chemistry, Faculty f Agriculture* and the Department f Bacterilgy, Sectin f Antibitic Research, Schl f Medicine**, Thku University, Sendai) (Received fr publicatin, April 15, 1969) Amin acids were shwn t react with flurescein-isthicyanate (FITC) t frm flurescein-thicarbamyl amin acids (FTC-amin acids). Flurescein-thicarbamyl amin acids were cnverted int flurescein-thihydantin amin acids (FTH-amin acids) under acidic cnditin. Amin acid flurescein-thihydantins were successfully separated by thin-layer chrmatgraphy. These results are discussed in cnnectin with the applicatin fr micr-analysis f amin acids, peptides and prteins. Varius methds are nw available fr use in structural studies f prteins and peptides. Amng them, the 2, 4-dinitrflurbenzene methd (1,2) and the dimethylaminnaphthalenesulfnyl chlride methd (3 5) are mst widely used fr chemical determinatin f the N-termini f peptides. These methds are, hwever, limited t the determinatin f nly the N-terminal residue and mre cmplicated strategies are required fr the sequential analysis f peptides. On the ther hand, phenylisthicyanate (6), which is cmmnly used fr the sequential analysis f peptides, needs relatively large quantities f peptides fr the direct identificatin f the derivatives. Otherwise the subtractive mdificatin is necessary (7). It is an advantage f ur methd ver knwn methds that it culd be applied t the sequential analysis f peptides and prteins with sensitivity higher than that in thers. Part I: J. Bichcm., 65, 777 (1969). t Present address: Children's Cancer Res. Fundatin, 35 Binney St. Bstn, Mass. 2115, U.S.A. In previus papers (8,9), it was reprted that FITC* was superir t ther reagents fr the N-terminus determinatin f prteins and plypeptides n a micr-scale. The N-terminal residues f insulin (1) and necarzinstatin (7/) were cnfirmed by using FITC. The FTH**-derivatives f the N-terminal amin acid residues were btained after the cleavage f the FTC-prteins with trifluracetic acid and they were identified by thin-layer chrmatgraphy (9). The reactin mechanism f FITC with prteins was described in a previus paper (9). In the present study FITC was allwed t react with twenty ne natural amin acids. FTC***-amin acids thus frmed were cnverted int their hydantin frms under acidic cnditins and were identified by thin-layer chrmatgraphy. It was pssible t demnstrate the cnversin f a FTC-amin acid int a * Fluiescein-isthicyanate. ** Flurescein-thihydantin ; Althugh the expressin f FTH-amin acid is incrrect, this was emplyed in this paper fr cnvenience. * Flurescein-thicarbamyl. 783 Dwnladed frm at Penn State University (Patern Lib) n May 8, 216

2 784 H. KAWAUCHI, K. TUZIMURA, H. MAEDA and N. ISHIDA FTH-amin acid with the aid f thin-layer chrmatgraphy and infrared spectrmetry, suggesting that the ver-all reactin mechanism (DIAGRAM 2) is similar t that fr Edman's phenyl isthicyanate. Using thin-layer chrmatgraphy technique it was pssible t separate and identify mst f the cmmn amin acids. Materials. EXPERIMENTAL Flurescein-isthicyanate I: Accrding t the methd f COONS and KAPLAN (12), nitrflurescein was prepared frm resrcinl and 4-nitrphthalic acid. Nitrflurescein I and II were separated frm each ther by fractinal crystallizatin f the reactin mixture. Aminflurescein I was btained by alkaline reductin f nitrflurescein I accrding t MCKINNEY et at. (13). Finally flurescein-isthicyanate I was prepared frm aminflurescein I by treatment with thiphsgene accrding t the methd f Ris et al. (14 ). The FITC-I was washed several times with 6N HCl at rm temperature and dried ver PJOJ in a vacuum desiccatr. The resulting HCl salt was range pwder and chrmatgraphically pure n thin-layer when bserved under ultravilet light. N-C-S Ismer I S=C=N VV II COO" Ismer II Structures f flurescein-isthicyanate ismer-i and II In ur studies, the ismer I was used because this ismer culd be prepared easily. The reactivity f the ismer II was nt studied. All slvents used fr the thin-layer chrmatgraphy were redistilled. Other materials used in this experiment were same as thse described in ur previus papers (8, 9). Methds. Preparatin f Flurescein-thihydantins f Amin Acids Flurcscein-isthicyanate was disslved in acetne with a trace amunt f pyridine, and the slutin was added t an amin acid slutin in carbnate buffer (see "RESULTS")- The reactin was stpped by acidifying the mixture with glacial acetic acid. In rder t facilitate the cyclizatin t yield the crrespnding thihydantin f the FTC-amin acid, 6 N HCl was added t give a final cncentratin f 2N HCl. The cyclizatin reactin was fllwed by using thin-layer chrmatgraphy (Fig. 3). A representative prcedure attained is described in the Amin acid slutin Acetne slutin f in.2 M carbnate + FITC-I with trace buffer ph9. pyridine at 2 C Acetic acid Acetne HCl at 2 C FTH-amin acid (acidify) 4hr (precipitate) (slubilize) (cyclize) 12 hr DIAORAM 1. Representative scheme f preparatin f FTH-amin acids. Measurement f Infrared Spectra The infrared absrptin spectra f FTC- and FTH-amin acids were recrded with an Infrared Spectrphtmeter, Mdel IR-S (JASCO), using a ptassium brmide disc (Fig. 4). Electrphresis Paper electrphrcsis f FTC-amin cm FITC Ala Ser 8V Trp u Glu FITC FIG. 1. Paper-electrphresis f fluresceinthicarbamyl amin acids; at a ptential gradient f lov/cm in.1 M Na-phsphate buffer (ph4.) fr 23min. m 5 Dwnladed frm at Penn State University (Patern Lib) n May 8, 216

3 Preparatin and TLG f FTH-Amin Acids 785 filter paper.1m Naacids was perfrmed n 17 cm x 3 cm (Ty N. 51) at 3 V, fr 3--thr i phsphate buffer (ph4.) (Fig. 1). Separatin f FTH-Amin Acids by Thin-layer Chrmatgraphy Thin-layer chrmatgraphy f FTH-amin acids was perfrmed in a jar (24x21 x5cm) n plates (.25 mm in thickness) f silica gel (Kiesel Gel G, Merck C.), which had been activated fr 6min at Slvent systems selected fr the present experiments were as fllws; A: Benzene: pyridine: acetic acid ( 6:1:1, v/v/v) B : Benzene: ethyl acetate: acetic acid ( 5: 1:1, v/v/v) C: Chlrfrm: ethanl: acetic acid (1:5:3, v/v/v) D: Chlrfrm: pyridine: acetic acid (15:3:1, v/v/v) E: Chlrfrm: pyridine: acetic acid (1:1:2, v/v/v) F : n-buthanl: acetic acid: water ( 4:1:1, v/v/v) FTH-amin acids used fr chrmatgraphy were taken directly frm the reactin mixture in the micrscale preparatin withut separatin and purificatin. RESULTS Preparatin f Flurescein-ihihydantin Amin Acids. Shwn belw is a representative prcedure fr the preparatin f FTH-amin acids. FTH-Phenylalanine One hundred and fifty mg f phenylalanine (.91 mmles) in 25 ml f.2 M carbnate-bicarbnate buffer (ph 9.) was allwed t react with 1 mg f FITC-I (.2 mmles) in 5 ml f acetne cntaining a trace amunt f pyridine at 25 C. The reactin was stpped after 4 hr by acidifing the mixture with glacial acetic acid t ph 4.5. The resulting precipitate f FTC-Phe was extracted with ethyl acetate and the extract was cncentrated in vacua t abut 5 ml. This cncentrate was applied t a silica gel clumn and eluted with chlrfrm: ethanl: acetic acid (16:2:1, v/v/v). The FTC-Phe fractin was cncentrated in vacua. This was disslved in 3 ml f acetne and allwed t cyclize after the additin f 5 ml f 6 N HC1 at 23 C. The reactin was stpped when FTC-Phe was n lnger detectable n a thin-layer chrmatgram. FTH-Phe thus prepared was purified with a silica gel clumn. The FTH-Phe fractin was cncentrated and FTH-Phe precipitated with a small vlume f.1 N HC1. Yield apprx. 6% based n FITC. Elemental analysis ; Calc. C: 67.15, H: 3.76 fr COHJON.O.S, Fund. C: 65.86, H: 4.63,[a] D =,mp: 3 C (decmp.). The preparatin was chrmatgraphically pure n thin-layer. Lw carbn cntent f elemental analysis was likely due t its hygrscpicity. Infrared spectra f FTC- and FTH-Phe are shwn in Fig. 4. Micr-scale Preparatin f FTH-Amin Acids fr Chrmatgraphy We studied the preparatin f standard FTH-amin acids n micr-scale fr rutine wrk f N-terminal analysis. Each amin acid in slutin (.5 ml; 1 ^mles) was mixed with the FITC-I slutin (.5 ml; 3 ^mles) in a small brwn test tube. The reactin was perfrmed with the same cnditins as described abve. The reactin mixture was acidified with glacial acetic acid and the precipitate frmed was disslved in abut.5 ml f acetne. T this slutin.2 ml f 6 N HC1 was added and the prduced FTC-amin acid was allwed t cyclize t yield FTH-amin acid. These FTH-amin acid slutins were stred at 4 C until use fr chrmatgraphy. : Flurcscein chrmphre -N=C=S (FITC-1) OH" R NH 2 -CH-COCT (Amin acid) R )-NH-C-NH-CH-COO" (FTC-Amin acid) (FTH-Amin acid) DIAGRAM 2 The reactin mechanism (DIAGRAM 2) was similar t that f EDMAN'S phenylisthicyanate methd (6,15). The thicarbamylatin reactin was affected by ph and temperature. It was best perfrmed with a minimum f Dwnladed frm at Penn State University (Patern Lib) n May 8, 216

4 786 H. KAWAUCHI, K. TUZIMURA, H. MAEDA and N. ISHIDA side reactins in.2 M sdium carbnate-bicarbnate buffer, ph 9., at 2 25 C. At lwer ph the reactin was slw and at higher ph side reactins ccurred. The majr side reactin abve ph 1 seemed t be a degradatin f FITC t aminflurescein, fr a flurescent spt fund at the rigin n thin-layer chrmatgram was identical t the authentic aminflurescein-hcl. Under the ptimum cnditins aliphatic and armatic amin acids reacted quickly with FITC-I (Fig. 2) but trifunctinal amin acids such as acidic r basic amin acids reacted relatively slwly in the micr-scale preparatin. FTH-amin acid slutins stred at 4 C were stable fr ne r tw weeks, except fr the derivatives f serine. threninc, glycine, asparagine, glutamine and prline. FTH-serine and -threnine shwed tw spts respectively n thin-layer. These new spts were prbably caused by the decmpsitin int the dehydr derivatives as shwn fr PTH-serine and -threnine {16). In the case f FTH-glutamine and -asparagine, minr spts were identified as FTH-glutamic acid and -aspartic acid respectively with authentic samples. I TIME OF REACTION ( hr) Fi. 2. Rate f reactin f FITC with alanine. Time curse study f cupling reactin f alanine with FITC at ph9., 23 C in.2 M carbnate buffer. The rate f reactin was measured by O.D. measurement at 495 m// f FTC-alanine after separatin by passage f an aliqut frm the reactin mixture thrugh a cellulse clumn with.2 M NajHPO, as the elucnt. Thin-layer Chrmatgraphy f FTH-Amin Acids. The samples f FTH-amin acid fr chrmatgraphy were taken directly frm the reactin mixture withut separatin r purificatin. Althugh small spts f unreacted reagent and decmpsitin prducts appeared n chrmatgrams, they were eaily distinguished frm the majr prducts. Unreacted FITC- I ran behind the slvent frnt and aminflurescein - HC1, a decmpsitin prduct, stayed at the rigin. Spts cntaining 1"' mle f FTC-amin acids r FTH-amin acids were detectable by their yellwish clr and spts cntaining 1" 1! mle were detectable by their yellw green flurescence under ultravilet light. All FTH-amin acids were mre easily detected when the thin-layer chrmatgrams were treated with ammnia gas. Flurescein-thihydantin amin acids f neutral and acidic amin acids were separated FTH- Amin acids Gly Ala Val Leu He Ser Thr Asp Glu CySO,H Met Phe Tyr Pr Hyp Trp Lys His Arg TABLE Siparatin f FTH-amin acids n thin-layer f silica gel. I Rf values in different slvent systems" A B C ; : ( D ) Cnditins are described in the text. E Dwnladed frm at Penn State University (Patern Lib) n May 8, 216

5 Preparatin and TLC f FTH-Amin Acids TABLE II 8 Rf values f FTH-basic amin acids and -CyS3H n thin-layer f silica gel. FTH-Amin acid System (Buthanl: acetic acid : water) 2:1:1 3:1:1 4:1: with the abve slvent system, as shwn in Table I. FTH-phenylalanine and -methinine were successfully separated with the slvent system chlrfrm : benzyl alchl: acetic acid (2:9: 1, v/v/v). In this case the.ft/values f FTH-phenylalanine and -methinine were.63 and.52 respectively. FTH-leucine and -isleucine were nt separated. FTH-basic amin acids did nt mve in any f the slvent systems mentined abve. n-buthanl: water: acetic acid (4:1:1, v/v/v) gave relatively gd reslutin fr these derivatives (Table II). The Rf value f an FTH-amin acid is very much affected by the vapr pressure f the develping slvent. Therefre the develping jar must be saturated enugh with the slvent vapr. FTC-amin acids were studied in the same manner as the FTH-amin acids. In shrt, the FTC-amin acids exhibited lwer mbility in the same chrmatgraphic systems. The Rf values f the FTC-amin acids were abut 6% f thse f the FTH-amin acids. DISCUSSION In the present paper we have described a methd fr preparatin and identificatin f FTH-amin acids. The present methd allws the identificatin f a very small amunt f amin acids n thin-layer chrmatgraphy and has a ptential fr use in sequence analysis as we described in the previus papers (8,9). The FTH- and FTC-amin acids are visible as yellw spts n thin-layer chrma- FITC 7 (J 6 5 FNH2 M-FTH-Al.- 4 tr: ^-FTC-Ala 3 A B C FNHg D E : ' FIG. 3. The frmatin f FTH-amin acid fllwed by thin-layer chrmatgraphy. The transfrmatin f a FTC- t a FTHamin acid is visualized in this figure. Hydrchlric acid treatment f FTC-Ala (D) was cmpared with acetic acid treatment (C). After hydrchlric acid treatment, all FTC-Ala has been cnverted int FTH-Ala by cyclizatjn. A: Aminflurescein I. B: Flurescein-thicarbamylatin reactin mixture. C: Frmatin f FTH-Ala in acidic slutin with acetic acid. D: Frmatin f FTH-Ala in acidic slutin with 2N HC1. E: Flurescein isthicyanate. Plate: Kiselgel G..25mm in thickness, Develper; Benzene : ethylacetate : acetic acid (5: 1 : 1, v/v/v), Detectin was made frm the yellw red clur and flurescence under ultravilet light. tgrams when each spt cntains mre than 1"8 mle and are visible as intense flurescent greenish yellw spts under ultravilet light when each spt cntains mre than 1"1* mle. The transfrmatin f FTC- t FTH-amin acids was described. Acid treatment f FTCamin acids facilitates the frmatin f FTHamin acids as bserved n thin-layer chrmatgraphy (Fig. 3). This cnversin was als cnfirmed by the infrared spectra (Fig. 4). Tw peaks were fund in FTC-phenylalanine, i.e. at 1,71 cm"1 and 1,59 cm"1. The peak at 1,71 cm"1 was due t the carbxyl grup f phenylalanine residue. The peak at 1,59 cm" 1 was due t the carbxyl grup f the flurescein chrmphre in the carbxylate frm. This absrptin at 1,59 cm" 1 was cmmnly bserved in flurescein derivatives studi- Dwnladed frm at Penn State University (Patern Lib) n May 8, 216 FTH-Lys FTH-Arg FTH-His FTH-CySOjH F-NH, FITC-I 787

6 788 H. KAWAUCHI, K. TUZIMURA, H. MAEDA and 1 JO* 9 rt I I ' I T4H9 18" 17 16!5 14 cm" 1 FIG. 4. Infrared spectrum f FTC- and FTH-phenylalanine at 1,9 1,4cm"1 regin. A: FTC-Phenylalanine in KBr disc. B: FTH-Phenylalanine in KBr disc. ed whereas the absrptin at 1,71 cm" 1 was absent in free flurescein. Thus the peak at 1,71 cm" 1 in FTC-phenylalanine was surely due t the carbxyl f phenylalanine. This absrptin disappeared when FTC-phenylalanine was cnverted int FTH-phenylalanine. This indicates the participatin f the carbxyl grup f phenylalanine residue in the frmatin f the thihydantin ring resulting in FTH-phenylalanine. A new strng brad band appeared at 1,745 cm"1 in the spectrum f FTH-phenylalanine. This was assigned t the carbnyl f the thihydantin ring. Other FTH-amin acids als had the peak at 1,76 1,73 cm"1. On the basis f this evidence, the mechanism f the reactin f FITC with amin acids, which is shwn in the DIAGRAM 2, has been prpsed. The behaviur f FTC- and FTH-amin acids n thin-layer chrmatgraphy was similar t that f PTC*- and PTH**-amin acids. FTC-amin acids migrated in abut 6% f the distance f crrespnding FTH-amin acids in mst f the slvent systems. In the present study, each f the seventeen mn amin mn carbxyl amin acids resulted in a single spt f FTC-amin acid n chrmatgraphy after the cndensatin and cyclizatin reactins. Glutamic and aspartic * Phenyl-thicarbamyl * Phenyl-thihydantin- acids, which are dicarbxyl amin acids, gave similar results. Serine and threnine, which cntain a hydrxyl grup, als shwed nly ne spt after the cndensatin reactin. Hwever, when they were treated with acid, tw spts appeared. This fact prbably shws that they decmpsed the FTH-dehydr-derivatives. Lysine, which bears tw amin grups, prduced mre than ne FTH-amin acid althugh the N-a-FTH-lysine was the majr prduct. The sulfhydryl grup f cystcine seemed t be reactive, because there were tw spts n the thin-layer chrmatgram befre cyclizatin. These tw spts had Rf values f and.5 respectively. Only ne spt appeared after acidificatin. This is because the sulfhydryl grup f cysteine was invlved in the reactin with FITC but hydrlysis f S-FTCcysteine ccurred in the acidic cnditin. Phenxy grup f tyrsine seemed t react t a lesser extent with FITC-I in the cnditin emplyed. The majr spt fr tyrsine seemed t be N-a-FTC-tyrsine. A minr spt might be a prduct resulting frm a reactin f the tyrsyl hydrxy grup. The islatin f this minr prduct has nt been attempted at this time. Other grups that were investigated briefly were the imidazle, indl, amide, guanidyl grups f histidine, tryptphan, glutamine, asparagine and arginine respectively. Mst f these yielded ne- majr spt as described abve. Histidine tends t prduce an extra minr spt n thin-layer chrmatgraphy. Separatin and identificatin f the FTHamin acids were described as shwn in Tables I and II. Mre sensitive identificatin was pssible by expsing the thin-layer plate t ammnia gas t bring it t alkaline ph after all the slvent was evaprated. The applicatin f this methd fr the sequence study f knwn and unknwn prteins and peptides is under way in ur labratries. Physicchemical studies f the FTH-amin acids will be reprted elsewhere. Dwnladed frm at Penn State University (Patern Lib) n May 8, 216 n» 9 i8d~17b N. ISHIDA

7 Preparatin and TLC f FTH-Amin Acids 789 We wish t thank Mr. Y. Nara fr his assistance in the thin-layer chrmatgraphy f FTH-amin acids and Mr. H. Kursu in the preparatin f fluresceinisthicyanate. REFERENCES ( 1 ) F. Sanger, Bichem. J., 39, 57 (1945) (2) H. Fraenkel-Cnrat, J.I. Harris, and A.L. Levy, "Methds f Bichemical Analysis," ed. by D. Glick, Interscience Pub. Inc., New Yrk, Vl.11, p. 359 (1955) (3) W.R. Gray and B.S. Hartley, Bichem. J., 89, 59p (1963) (4) W.R. Gray and B.S. Hartley, Bichem. J., 89, 379 (1963) (5) W.R. Gray, "Methds in Enzymlgy," ed. by C.H.W. Hirs, Academic Press Inc., New Yrk, Vl. XI, p. 139 (1967) (6") P. Edman, Acta Chtm. Scand., 4, 283 (195) ( 7 ) W. Knigsberg, " Methds in Enzymlgy," ed. by C.H. Hirs, Academic Press Inc., New Yrk, Vl. XI, p.461 (1967) (8) H. Maeda and H. Kawauchi, Bichem. Biphys. Res. Cmmun., 31, 188 (1968) (5) H. Maeda, N. Ishida, H. Kawauchi, and K. Tuzimura, J. Bichem., 65, 777 (1969) (7) F. Sanger, Bichem. J., 44, 126 (1949) (11) H. Maeda, K. Kumagai, and N. Ishida, J. Antibitic Ser. A, 19, 253 (1966) (12) A.H. Cns and M.H. Kaplan, J. Exptal. Med., 91, 1 (195) (13) R.M. McKinney, J.T. Spillane, and G.W. Pearce, J. Org. Chem., 27, 3986 (1962) (14) J.L. Riggs, R.J. Seiwald, and J.H. Burckhalten, Am. J. Pathlgy, 34, 181 (1958) (15) P. Edman, Acta Chtm. Scand., 1, 761 (1956) (16) A.L. Levy and D. Chung, Bichim. Biphys. Acta, 17, 454 (1955) Dwnladed frm at Penn State University (Patern Lib) n May 8, 216