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Ion-pair high-performance liquid chromatography of the insect neuropeptide proctolin and some analogs
id of Chramutogmphy, 194 (1980) 421423 Ebvier t3eicnm PubF.Ming Company, Amsterdam -
printed in The Nictherlands
ht-pair high-performance liquid chromatography of the insect neurepeptide proctulin and sume anatogs ALVIN N. STARRATP and MARY E STEVENS . Agncuhm canedn. I%esemch Iirstitufe,UniversitySub Post O-e,
London, Oz&rio N6A 5B7 (Cam&)
(Receivfxl February 26t4 1980) Aithough the potentinsectrnyotropicpeptideproctolin,Arg-Tyr-Leu-Pro-Thr, has heen characterizedby a number of methods includingpaper and thin-layerchromatography, high-voItage paper electrophoresisand pharmacological assay’*2, a rapid, unambiguous means of identiiicationand purity assessment has not been available. Recently,Hohan and Cooks-+ have reportedshe analysis of proctolin in insect extracts by high-performanceliquid chromatography(HPLC) of the phenylthiocarbamylderivative.In this paperwe reporton the use of ion-pair reversed-phase HPLCY for the anaIysis and characterizationof underivatizedproctolin and its separationfrom some closely relatedanalogs. EXPERJMENTAL
Reversed-phaseHPLC was carriedout on a 30 cm x 3.9 nun I.D. pBondapak fatty acid analysis cohmn (Waters Assoc., Milford, MA, U.S.A.) using a Waters liqtid chromatographic system equipped with a Model 6OQOApump, a Model USK sample tijector and a SchoefIel Model SF-770 variable-wavelengthdetector operated at 225 nrn. All chromatography was performed at room temperature. Distilled water and HPLC grade acetotitri!e (C&don Labs., Georgetown, Canada} wefe used for the mobile phases. Ion-pairing reagents used were PIG Reagent B-6 obtained from Waters Assoc., trifiuoroaceticacid (JTFA) from l%stmm-Kodak (Rockter, NY, US-A& heptafluorobutyricacid (HFBA) from P&&z aad Bauer (Sta&ord, CT, U.S.A.) arid orthophosphoric acid and trichloroaceticacid @CA) from Far Scientific(Toronto, Canada). Before degassingand me, the ace&o&rile was filteredthrongh a Miilipore FHUP OS&pm Glterand the water containing the ion-pairing reagent was I%xed through a _MiBiporeHAWP 0_45-pm Glter. The cohmn was equilibratedto new solvents for at least 30 min. The pentapeptides,synthesizedand purifiedin this laboratory by standard pOXdm’ms, were dissolvedin distilledwater (I &,ul) and l-10 ~1 were injected_
Proctolin was separatedfrom analogs differingby only a single amino acid by ion-pair n%zrsed-phaseHPLC on a pI3ondapa.kfaety acid amlysis column using
NOTES
422
acetonitril~water mixtures (Iable I). Marked alteretioas in retedon tins we= achieved by varying the ion-pairing reagent. In agreementwith previous studies of small wptides* the order of eiution paralleledthe hydrophobicityof the constituent ~-amino acids. Similarresultswereobtainedwhen 2 ~Boudapak C& column was used. TABLE I EFFECT Abii
OF ION-PAIRING
SOME
REJATED
REAGEENTS ON THE RETENTION
PENTAPEFI-IDES
ON
A pRONDAFAK
TIMES OF PROCTOLIN FATTY ACID ANAI_YSTS
COLUMN Retentror: time (m&E)
PI@&?
i-&PO,’
Arg-Tyr-Jieu-Ro-Thr h-g-Ty-r-Leu-Ro-Ala &g-Tyr-Leu-Ah-Thr .4sg-TywUa-R+Thr A.ii-Ala-LedJro-Thr Ala-TydLeu-Ro-Thr kg-Tyr-LeM?ro-D-Thr Ars_ryr-LewD-Ro=i%r Jug-Tyr-o-b-bThr kg-~-Tyr-terr-P E-Arg-Tyr-Leu-FmThr Arg-Tyr-Leu-Fro&x Arg-Fix-Leu-Ro-Thr mS r&f
7.1 7-2 4.6 3.1
125 10.2 9.7 16.5 21.7 14.3 5.9 15.0
in acetonitde-water
-.. 5 m&f ic acctodrik-ws_er
TFA ‘
Il.3 176 7.0 4.3 52 16.4 17s 16.1 24.8 9.3
TFA”
3.5 3.7 3.0 2.4 L6 4.0 3.8 4.1 5.5 5-T 4.5 3.0 5.3
TCA”
5.5 6.1 4.1 3.3 3.6 5.2 6-7 1::: IO.5 8.4 4.8 10.1
HFBA”
8.2 9.4 69 4.5 5.2 5.4 10.1 112 14.8 IS.9 12.7 73 16.1
PK Reagent Bd”
Zibdzim iiermre
SdfonQzc-
S-i 5.7 44 3.4
3.7 3.9 3-4 27
4-4 5.8 S-7 8.0 8.7 7.2 4.5 8.5
2.4 4.6 4-6 7.4 5.7 4.7
(1:9); flow-rate 1.5 td/min. (1 r4); flow-rati I.5 m&lin_
Proctolinand the five possiblediastereoisomersresultingfrom the replacement of ezh of the amino acids in turn with the respective~-amino acid could also easily be separated.The diestereoisomers;ttlelutedmore slowly than proctolinwith highest hydrophobicitybeing found for @-Ty$j-proctolin. Separationof peptidediastereoisomershas previouslybeen diss in termsof conformationaldifferencesbetween the isomerslelz_ Ali the peptideseluted more slowIy with PIG Reagent B-6 (5 m&4hexaue sulfonic 2cid containing suflicientaceticacid to yieid a pH of about 3.5) than with 5 m&f sodium hexane sulfonate @H 6.4) due to suppressionof ionization of the carboxyl group and more el%ctlve ion-pairingwith *&ea-amino group at the lower pII. HFBA, which to our knowledgehas not been reportedpreviouslyas an ionpairingrcqmt, gave excellentresolutionof proctolinand most of its analogs. Fig. I iIllXtE%tes ?X!SUltS Witi this reagent. Slight inin the capacity fers and SIII& changer in the separationfactors for the peptideswere observed when the amount of HFBA was in-4 from 5 mM to 25 m&f. Like TFA”, I-IFBA may be readily removed by lyophilization so systems containing this reagent should be nsefnI for preparativechromatographyof peptides. Our resultsfurtherilhrstratethe utilityof ion-pair reversed-phaseHPLC using isoczaticsystemsin separatingand characterizingsmah, underivatizd, cIoseIyreiated
423
4
8
12
;
Time (minutes) pi I_ ~lution patpem of pmctoli and five analos on a &3ondapak fatty acid analysis column with ~~~t~nitde-water (I :4) containing 5 rnM HFBA as eluent at a fiow-rate of I.5 mlbi~. Peaks: 1 = ~~3~roaolin; 2 = fAlalf-proctok; 3 = [Ala‘&proctolin; 4 = proctoIin; 5 = [Alas]-prwtoI& 6 = zp’ne21_proctolin.
peptides.More specificallythey indicatethat this techniquewill be very convenientfor determiningthe homogeneity of syntheticproctolin and its analogs, including the detection of diastereoisomericimpurities,as well as for monitoring the stability of solutions of these peptides. It is also anticipated that ion-pair reversed-phase HPLC can be empIoyed to simplify the lengthy isolation procedurefor proctolirP and, by se-kg proctolin from other insect myogenic peptides, will permit the bioassay of low levels of proctolin in relativelycrude extracts preparedfrom either whole insectsor specik tissues. REFERENCES 1 B. E. Brown+ Ufe ski., 17 (1975) 1241. 2 A. N. Ste_;att and B. E Brovn, Ufe sci., 17 (1975) 1253. 3 G. M. Human and B. J. Cook, Camp Bfodiem. Physiil., 62C (1979) 231. 4 G. M. Ko?martand B_ J. Gmk, hsect Biochem, 9 (1979) 149. 5 E Tosdinsoq T. M. J&cries and C. M. Riley, 1. Chromatogr., 159 (1978) 315. 6 M. T. W. Eieasn and W. S. Hancock, Trends B&diem. Ski., 4 (1979) NS8_ 7 A. N. Starrattand B. E Broyn, CQIL1. Chem., 5.5(1977) 4238. 8 A. N. Soarrattand & E Brown, Bid&em. siophys RES_ CORWUZ., 90 (1979) ins. 9 M. 3. O’Hare and E. C. Nice, J. Ckro~~:ogr., 171(1979) 209. 10 E. P. KraeiTand D. J. Fietrzyk, Anal, Chema, 50 (1978) 1353. 11 B. hrsen, B. L. Fox, M. F. Burke and V. J. Hruby, Inf. J. Peptide Protein Res., 13 (1979) 12. 12 S. Tefabc, R. Ksxak.a and K. Inouye, J_ Chvnrtzroogr_, 172(1979) 163. 13 C. E Dinlap II& S. Gentiernan and L. I. Luwney, J_ Chrumdogr., 168(1978) 191. 14 B_ E. Brown and A. N. Starrat&J. lirrect Phrysiol.,21(1975) 1879.
printed in The Nictherlands
ht-pair high-performance liquid chromatography of the insect neurepeptide proctulin and sume anatogs ALVIN N. STARRATP and MARY E STEVENS . Agncuhm canedn. I%esemch Iirstitufe,UniversitySub Post O-e,
London, Oz&rio N6A 5B7 (Cam&)
(Receivfxl February 26t4 1980) Aithough the potentinsectrnyotropicpeptideproctolin,Arg-Tyr-Leu-Pro-Thr, has heen characterizedby a number of methods includingpaper and thin-layerchromatography, high-voItage paper electrophoresisand pharmacological assay’*2, a rapid, unambiguous means of identiiicationand purity assessment has not been available. Recently,Hohan and Cooks-+ have reportedshe analysis of proctolin in insect extracts by high-performanceliquid chromatography(HPLC) of the phenylthiocarbamylderivative.In this paperwe reporton the use of ion-pair reversed-phase HPLCY for the anaIysis and characterizationof underivatizedproctolin and its separationfrom some closely relatedanalogs. EXPERJMENTAL
Reversed-phaseHPLC was carriedout on a 30 cm x 3.9 nun I.D. pBondapak fatty acid analysis cohmn (Waters Assoc., Milford, MA, U.S.A.) using a Waters liqtid chromatographic system equipped with a Model 6OQOApump, a Model USK sample tijector and a SchoefIel Model SF-770 variable-wavelengthdetector operated at 225 nrn. All chromatography was performed at room temperature. Distilled water and HPLC grade acetotitri!e (C&don Labs., Georgetown, Canada} wefe used for the mobile phases. Ion-pairing reagents used were PIG Reagent B-6 obtained from Waters Assoc., trifiuoroaceticacid (JTFA) from l%stmm-Kodak (Rockter, NY, US-A& heptafluorobutyricacid (HFBA) from P&&z aad Bauer (Sta&ord, CT, U.S.A.) arid orthophosphoric acid and trichloroaceticacid @CA) from Far Scientific(Toronto, Canada). Before degassingand me, the ace&o&rile was filteredthrongh a Miilipore FHUP OS&pm Glterand the water containing the ion-pairing reagent was I%xed through a _MiBiporeHAWP 0_45-pm Glter. The cohmn was equilibratedto new solvents for at least 30 min. The pentapeptides,synthesizedand purifiedin this laboratory by standard pOXdm’ms, were dissolvedin distilledwater (I &,ul) and l-10 ~1 were injected_
Proctolin was separatedfrom analogs differingby only a single amino acid by ion-pair n%zrsed-phaseHPLC on a pI3ondapa.kfaety acid amlysis column using
NOTES
422
acetonitril~water mixtures (Iable I). Marked alteretioas in retedon tins we= achieved by varying the ion-pairing reagent. In agreementwith previous studies of small wptides* the order of eiution paralleledthe hydrophobicityof the constituent ~-amino acids. Similarresultswereobtainedwhen 2 ~Boudapak C& column was used. TABLE I EFFECT Abii
OF ION-PAIRING
SOME
REJATED
REAGEENTS ON THE RETENTION
PENTAPEFI-IDES
ON
A pRONDAFAK
TIMES OF PROCTOLIN FATTY ACID ANAI_YSTS
COLUMN Retentror: time (m&E)
PI@&?
i-&PO,’
Arg-Tyr-Jieu-Ro-Thr h-g-Ty-r-Leu-Ro-Ala &g-Tyr-Leu-Ah-Thr .4sg-TywUa-R+Thr A.ii-Ala-LedJro-Thr Ala-TydLeu-Ro-Thr kg-Tyr-LeM?ro-D-Thr Ars_ryr-LewD-Ro=i%r Jug-Tyr-o-b-bThr kg-~-Tyr-terr-P E-Arg-Tyr-Leu-FmThr Arg-Tyr-Leu-Fro&x Arg-Fix-Leu-Ro-Thr mS r&f
7.1 7-2 4.6 3.1
125 10.2 9.7 16.5 21.7 14.3 5.9 15.0
in acetonitde-water
-.. 5 m&f ic acctodrik-ws_er
TFA ‘
Il.3 176 7.0 4.3 52 16.4 17s 16.1 24.8 9.3
TFA”
3.5 3.7 3.0 2.4 L6 4.0 3.8 4.1 5.5 5-T 4.5 3.0 5.3
TCA”
5.5 6.1 4.1 3.3 3.6 5.2 6-7 1::: IO.5 8.4 4.8 10.1
HFBA”
8.2 9.4 69 4.5 5.2 5.4 10.1 112 14.8 IS.9 12.7 73 16.1
PK Reagent Bd”
Zibdzim iiermre
SdfonQzc-
S-i 5.7 44 3.4
3.7 3.9 3-4 27
4-4 5.8 S-7 8.0 8.7 7.2 4.5 8.5
2.4 4.6 4-6 7.4 5.7 4.7
(1:9); flow-rate 1.5 td/min. (1 r4); flow-rati I.5 m&lin_
Proctolinand the five possiblediastereoisomersresultingfrom the replacement of ezh of the amino acids in turn with the respective~-amino acid could also easily be separated.The diestereoisomers;ttlelutedmore slowly than proctolinwith highest hydrophobicitybeing found for @-Ty$j-proctolin. Separationof peptidediastereoisomershas previouslybeen diss in termsof conformationaldifferencesbetween the isomerslelz_ Ali the peptideseluted more slowIy with PIG Reagent B-6 (5 m&4hexaue sulfonic 2cid containing suflicientaceticacid to yieid a pH of about 3.5) than with 5 m&f sodium hexane sulfonate @H 6.4) due to suppressionof ionization of the carboxyl group and more el%ctlve ion-pairingwith *&ea-amino group at the lower pII. HFBA, which to our knowledgehas not been reportedpreviouslyas an ionpairingrcqmt, gave excellentresolutionof proctolinand most of its analogs. Fig. I iIllXtE%tes ?X!SUltS Witi this reagent. Slight inin the capacity fers and SIII& changer in the separationfactors for the peptideswere observed when the amount of HFBA was in-4 from 5 mM to 25 m&f. Like TFA”, I-IFBA may be readily removed by lyophilization so systems containing this reagent should be nsefnI for preparativechromatographyof peptides. Our resultsfurtherilhrstratethe utilityof ion-pair reversed-phaseHPLC using isoczaticsystemsin separatingand characterizingsmah, underivatizd, cIoseIyreiated
423
4
8
12
;
Time (minutes) pi I_ ~lution patpem of pmctoli and five analos on a &3ondapak fatty acid analysis column with ~~~t~nitde-water (I :4) containing 5 rnM HFBA as eluent at a fiow-rate of I.5 mlbi~. Peaks: 1 = ~~3~roaolin; 2 = fAlalf-proctok; 3 = [Ala‘&proctolin; 4 = proctoIin; 5 = [Alas]-prwtoI& 6 = zp’ne21_proctolin.
peptides.More specificallythey indicatethat this techniquewill be very convenientfor determiningthe homogeneity of syntheticproctolin and its analogs, including the detection of diastereoisomericimpurities,as well as for monitoring the stability of solutions of these peptides. It is also anticipated that ion-pair reversed-phase HPLC can be empIoyed to simplify the lengthy isolation procedurefor proctolirP and, by se-kg proctolin from other insect myogenic peptides, will permit the bioassay of low levels of proctolin in relativelycrude extracts preparedfrom either whole insectsor specik tissues. REFERENCES 1 B. E. Brown+ Ufe ski., 17 (1975) 1241. 2 A. N. Ste_;att and B. E Brovn, Ufe sci., 17 (1975) 1253. 3 G. M. Human and B. J. Cook, Camp Bfodiem. Physiil., 62C (1979) 231. 4 G. M. Ko?martand B_ J. Gmk, hsect Biochem, 9 (1979) 149. 5 E Tosdinsoq T. M. J&cries and C. M. Riley, 1. Chromatogr., 159 (1978) 315. 6 M. T. W. Eieasn and W. S. Hancock, Trends B&diem. Ski., 4 (1979) NS8_ 7 A. N. Starrattand B. E Broyn, CQIL1. Chem., 5.5(1977) 4238. 8 A. N. Soarrattand & E Brown, Bid&em. siophys RES_ CORWUZ., 90 (1979) ins. 9 M. 3. O’Hare and E. C. Nice, J. Ckro~~:ogr., 171(1979) 209. 10 E. P. KraeiTand D. J. Fietrzyk, Anal, Chema, 50 (1978) 1353. 11 B. hrsen, B. L. Fox, M. F. Burke and V. J. Hruby, Inf. J. Peptide Protein Res., 13 (1979) 12. 12 S. Tefabc, R. Ksxak.a and K. Inouye, J_ Chvnrtzroogr_, 172(1979) 163. 13 C. E Dinlap II& S. Gentiernan and L. I. Luwney, J_ Chrumdogr., 168(1978) 191. 14 B_ E. Brown and A. N. Starrat&J. lirrect Phrysiol.,21(1975) 1879.