research article synthetic covalently linked dimeric form

Research ArticleSynthetic Covalently Linked Dimeric Form of H2 RelaxinRetains Native RXFP1 Activity and Has Improved In VitroSerum Stability

Vinojini B Nair12 Ross A D Bathgate13 Frances Separovic2 Chrishan S Samuel4

Mohammed Akhter Hossain12 and John D Wade12

1Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville VIC 3010 Australia2School of Chemistry University of Melbourne Parkville VIC 3010 Australia3Department of Biochemistry and Molecular Biology University of Melbourne Parkville VIC 3010 Australia4Department of Pharmacology Monash University Clayton VIC 3800 Australia

Correspondence should be addressed to Mohammed Akhter Hossain akhterhossainfloreyeduau and John D Wadejohnwadefloreyeduau

Received 9 September 2014 Accepted 11 October 2014

Academic Editor Andrei Surguchov

Copyright copy 2015 Vinojini B Nair et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles includingregulation of connective tissue remodeling and systemic and renal vasodilationThese effects aremediated through interactionwithits cognate G-protein-coupled receptor RXFP1 H2 relaxin recently passed Phase III clinical trials for the treatment of congestiveheart failure However its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance To increaseits residence time a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chainsincluding a judiciously monoalkyne sited B-chain followed by their combination through regioselective disulfide bond formationUse of a bisazido PEG

7linker and ldquoclickrdquo chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered The

resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1equally well It had fewer propensities to activate RXFP2 the receptor for the related insulin-like peptide 3 In human serum thedimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization maybe a viable strategy for producing longer acting variants of H2 relaxin

1 Introduction

Relaxin one of the first hormones to be discovered is amember of the insulin superfamily of peptides [1] It is asmall two-chain three-disulfide bonded peptide [2 3] Oncemuch ignored by the international research community [4]relaxin similar to its sister hormones insulin and the insulin-like growth factors (IGFs) is now known as amultifunctionalhormone It is primarily involved with the maintenance ofreproduction and pregnancy and in the facilitation of thedelivery of the young Its native G-protein-coupled receptorrelaxin family peptide receptor 1 [5] RXFP1 (previouslyknown as LGR7) was shown to be widely distributed invarious organs in both males and females Human (H2)

relaxin the major stored and circulating form of humanrelaxin is now known to play a key role in inflammatory andmatrix remodeling processes and possesses potent vasodila-tory angiogenic and other cardioprotective actions [6 7]

At physiological concentrations the H2 relaxin existsas a monomer [3] However Eigenbrot et al reported thecrystal structure for H2 relaxin and showed that it exists as anoncovalent dimer [8]This was confirmed by sedimentationequilibrium analytical ultra-centrifugation studies [9] Thisprobably corresponds to the stored form of H2 relaxin andsuch a dimer is likely to be biologically inactive because theknown key receptor binding residues (RB13 RB17 and IB20) inH2 relaxin [2 7] form part of the dimer interface [8] This issupported by the fact that themonomer of the related peptide

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 731852 9 pageshttpdxdoiorg1011552015731852

2 BioMed Research International

insulin is involved in its tyrosine kinase receptor activationwhereas its dimeric and hexameric forms are involved instabilizing the molecule during storage [10 11] A covalentlylinked (through a disulfide bridge) dimeric insulin peptidewas recently prepared by recombinant DNA technology Itneither bound to the insulin receptor nor induced ametabolicresponse in vitro [12] which is consistent with the view thatthe monomeric form is the active form of insulin Howeverthe insulin dimer was shown to be extremely thermodynam-ically stable in vitro which highlighted the importance ofoligomerization for insulin stability [12] This suggests thata covalently linked dimeric relaxin may also be more stablein vitro as well as in vivo compared to its monomeric formSuch a compound if it retained biological activity would bevery valuable given that H2 relaxin recently passed PhaseIII clinical trials for treating acute heart failure [13] despitehaving a short in vivo half-life of approximately 10min [14 15]that is characteristic of many peptides and proteins It is forthis reason that H2 relaxin requires continuous intravenousinfusion into patients over 48 hours [13] Therefore there isa clear need for improving the pharmacokinetic propertiesof H2 relaxin in order to potentially improve its therapeuticvalue

In this study we undertook to design and develop a cova-lently linked dimeric analogue of synthetic H2 relaxin usingboth click chemistry and a small polyethylene glycol (PEG)spacer to link the two monomers in such a structural orien-tation so as to retain biological activity (Figure 1) We studiedits structural and functional role using circular dichroism(CD) spectroscopy and RXFP1- and RXFP2-expressing cellsrespectively It was shown that the dimeric H2 relaxinpossessed a high degree of secondary structural similarityto native H2 relaxin Importantly unlike the insulin dimerthe dimeric H2 relaxin is equipotent to native H2 relaxinmonomer and exhibits improved in vitro serum stability

2 Experimental Procedures

21 Materials 9-Fluorenylmethoxycarbonyl- (Fmoc-) pro-tected L-120572-amino acids and 1-[bis(dimethylamino)methy-lene]-1H-benzotriazolium hexafluorophosphate 3-oxide(HBTU) were purchased from GL Biochem (ShanghaiChina) NN-Dimethylformamide (DMF) piperidine andtrifluoroacetic acid (TFA) were obtained from Auspep(Melbourne Australia) PAL-PEG-PS resins with substitu-tion of ca 020mmolg were purchased from AppliedBiosystems Inc (Melbourne Australia) Diethyl ethermethanol acetonitrile and dichloromethane were purchasedfrom Merck (Melbourne Australia) trifluoromethanes-ulfonic acid (TFMSA) anisole triisopropylsilane (TIPS) 36-dioxa-18-octanedithiol (DODT) and diisopropylethylamine(DIEA) were from Sigma-Aldrich (Sydney Australia)and 221015840-dipyridyl disulfide (DPDS) was from Fluka(Switzerland) CaCl

2was purchased from Mallinckrodt

(Melbourne Australia) and Na2HPO4and MgSO

4were pur-

chased from BDH Chemicals while KH2PO4

andMnCl

2were purchased from Ajax Chemicals (Sydney

Australia) Dulbeccorsquos modified Eagle medium (DMEM)penicillinstreptomycin L-glutamine and fetal bovine serum

were purchased from Invitrogen (Melbourne Australia)Sterile-filtered serum from human AB plasma was purchasedfrom Sigma-Aldrich (Sydney Australia) All other reagentswere purchased from Sigma-Aldrich (Sydney Australia)Native recombinant DNA-derived human relaxin-2 used ascontrol was obtained from Corthera Inc (San Francisco CAa subsidiary of Novartis AG Basel Switzerland) Syntheticeuropium-labelled H2 relaxin unlabelled H2 and humanINSL3 were prepared in-house as previously described[16 17]

22 Synthesis

221 Chemical Peptide Synthesis The H2 relaxin A- and B-chains were assembled as C-terminal amides on an auto-mated Protein Technologies Tribute peptide synthesizer(Tuscan AZ) or CEM Liberty microwave peptide synthesizer(Ai Scientific Australia) using Fmoc chemistry Side chainprotecting groups of trifunctional amino acids were TFA-labile except for tert-butyl- (tBu-) protected cysteine inposition A11 and acetamidomethyl- (Acm-) protected cys-teines in positions A24 and B23 Using instrument defaultprotocols both A- and B-chains were separately synthesizedeither at 01mmol scale or 02mmol scale activated witheither 4- or 5-fold molar excess of HBTU (04 or 05mmolfor a 01mmol scale of resin 08mmol for a 02mmol scaleof resin) in the presence of 5 equivalents DIEA Resin-attached peptides were treated with 20 vv piperidineDMFto remove N120572-Fmoc protecting groups When using themicrowave synthesizer coupling and deprotection steps werecarried out at 75∘C and 25W microwave power for 5minsand 60W microwave power for 3mins respectively whenusing the CEM Liberty microwave synthesizer Couplingand deprotection steps were carried out for 30 and 10minsrespectively when using Tribute synthesizers Upon completecoupling of the final amino acid of the native B-chain peptidesequence an extra amino acid containing the alkyne group(Fmoc-L-propargylglycine) was coupled by manual couplingprocedures

222 Peptide-Resin Cleavage Upon completion of solidphase synthesis the A- and B-chains were cleaved fromthe solid supports by treatment with TFA containinganisoleTIPSDODT (9425215 20mL) for 2 hCleaved products were concentrated byN

2bubbling precipi-

tated with ice-cold diethyl ether and centrifuged at 3000 rpmfor 5mins The centrifuged pellet was then washed with ice-cold diethyl ether and centrifugedThis process was repeatedat least three times

223 Peptide Purification All RP-HPLC analytical andpurification reactions were monitored using analytical andpreparativeVydac C18 columns (pore size 300 A particle size46times 250mmor 22times 250mm resp) in a gradientmodewitheluant A 01 aq TFA and eluant B 01TFA in acetonitrile

224 Preparation of Monoalkyne H2 Relaxin Followingsimultaneous cleavage and side chain deprotection and

BioMed Research International 3

ZLYSALANKCCHVGCTKRSLARFC

DSWMEEVIKLCGRELVRAQLAICGMSTWS



NN N

N

N

N

(PEG)7

NH2

H2N

O

O

(a)

0 10 20 30

1520

Time (min)

(b)

6000 10000 14000 18000

1255362

mz

(c)

Figure 1 (a) Primary structure and (b) RP-HPLC and (c) MALDI-TOFMS traces of synthetic H2 (PEG)7H2 dimer Theoretical [M + Na]+

125300

purification of the crude A- and B-chains stepwise formationof the three disulfide bondswas carried out via successive oxi-dation thiolysis and iodolysis as previously described [18]

225 Formation of H2 (PEG)7H2 Dimer PEG

7bisazide

(Jena Bioscience Germany) was coupled onto alkyne H2relaxin employing copper-catalyzed click reaction Thealkyne peptide was first dissolved in phosphate buffer saline(PBS) at pH 70 Following this 05 equivalents of PEG

7

bisazide and 20 equivalents of aqueous copper (II) sulfatepentahydrate were added sequentially Finally 21 equivalentsof aqueous ascorbic acid (made up fresh for use) wereadded and reaction left stirring at room temperature Thisreaction progress was monitored by analytical RP-HPLC andstopped after 45 minutes by diluting with approximately100 120583L deionised water before RP-HPLC purification

226 Peptide Characterization by MALDI-TOF MS Syn-thetic peptides (from intermediate steps and final product)were confirmed by matrix-assisted laser desorption ioniza-tion time-of-flight mass spectrometry (MALDI-TOF MS)utilizing a Bruker Ultraflex II instrument (Bruker Daltonics

Bremen Germany) using sinapinic acid (35-dimethoxy-4-hydroxycinnamic acid) matrix This matrix was made up in70 acetonitrile containing 01TFA

227 Peptide Quantitation by Amino Acid Analysis Peptidecontent and amino acid composition were determined usingvapour phase acid hydrolysis in 6M HCl2 phenol at 110∘Cfor 24 h Individual amino acids were converted to stablefluorescent derivatives using Waters AccQTag kit (WatersSydney Australia) Derivatized amino acids were separatedusing Shim-Pak XR-ODS (3 times 75mm 22 120583m) on ShimadzuRP-HPLC system (Shimadzu Victoria Australia)

228 Secondary Structure Analysis by CD Spectroscopy AJASCO instrument (J-185 Tokyo Japan) was utilized toobtain CD spectral analysis of all peptides The followingsettings were used to obtain readings wavelength range 195to 250 nm scanning speed 50 nmmin bandwidth 01 nmand cell length 1mm at 25∘C The peptide samples wereprepared at 02 120583g120583L in 10mM PBS pH 74 Raw data fromthe spectra in millidegree of ellipticity (120579) were converted tomean residual weight ellipticity (MRE) [19]


229 Receptor Binding Assays HEK-293T cells stably trans-fected with RXFP1 grown in DMEM medium supplementedwith 10FBS 100120583gmL penicillin 100120583gmL streptomycinand 2mM L-glutamine were plated out at 40 000 cells perwell per 200120583L in a 96-well ViewPlate with clear bottomand white walls precoated with poly-L-lysine Competitionbinding experiments were performed with 5 nM europium-labelled H2 relaxin [17] in the absence or presence ofincreasing concentrations of unlabelled peptidesNonspecificbindingwas determinedwith an excess of unlabelled peptides(500 nMH2 relaxin) Fluorescentmeasurementswere carriedout at excitation of 340 nm and emission of 614 nm on aVictor Plate reader (Perkin-Elmer Melbourne Australia)All data are presented as the mean plusmn SE of the totalspecific binding percentage (in triplicate wells) repeated in atleast three independent experiments and curves fitted usingone-site binding curves in GraphPad Prism 5 (GraphPadInc San Diego CA) Statistical differences in pIC50 valueswere analyzed using one-way analysis of variance (ANOVA)coupled to a Newman-Keuls multiple comparison test formultiple group comparisons in GraphPad Prism 5

2210 Functional cAMP Assay The influence of cAMPsignaling by synthetic analogues in HEK-293T cells express-ing either human RXFP1 or RXFP2 receptors was assessedusing cAMP reporter gene assay as described previously[20] Briefly HEK-293T cotransfected with response elementpCRE 120573-galactosidase reporter plasmid were plated outin Corning Cell BIND 96-well plate at 50 000 cells perwell per 200120583L 24 h later cotransfected cells were treatedwith increasing concentrations of H2 relaxin analogues inparallel with native H2 relaxin or human INSL3 After 6 hincubation at 37∘C cell media were aspirated and cells werefrozen at minus80∘C overnight A 120573-galactosidase colorimetricassay measuring absorbance at 570 nm on Benchmark Plusmicroplate spectrophotometer (Bio-Rad Gladesville Aus-tralia) was then used to measure relative cAMP responsesEach concentration point wasmeasured in triplicate and eachexperiment performed independently at least three timesLigand-induced stimulation of cAMP was expressed as apercentage of the maximum H2 relaxin response for RXFP1cells or human INSL3 for RXFP2 cells GraphPad Prism 5wasused to analyze the cAMP activity assay which was expressedas the mean plusmn SEM Statistical analysis was conducted usingone-way ANOVA with Newman-Keuls post hoc analysis

2211 In Vitro Serum Stability The stability of the syntheticanalogue was measured against native H2 relaxin in humanserum The purchased serum was not heat inactivated inorder to retain as much of its proteolytic enzymic activity aspossible The peptides tested were normalized to a final con-centration of 10mgmL with deionized water and 100 120583L ofpeptideswas added to 590 120583L 100human serumAfter addi-tion of peptide into the serum 50120583L of samples was removedand quenched with 250 120583L of ice-cold acetonitrile01 TFAThe solution was then spun down with a benchtop centrifugeat 13500 rpm (Eppendorf Centrifuge 5804R MelbourneAustralia) for 15mins at 4∘C to pellet the larger precipitated

serum proteins The remaining peptideserum solution wasplaced immediately into 37∘C incubator Degradation ofpeptides was monitored with manual RP-HPLC injectionsof supernatant using a Phenomenex Aeris Widepore 36 120583mC4 analytical column (pore size 100 A particle size 46times 250mm) in a stabilized gradient mode with eluent A01 aq TFA and eluant B 01TFA in acetonitrile Sampleswere taken at time points 05 10 15 20 25 3 4 5and 6 h Each assay was carried out in triplicate Elutionof target peptide was elucidated by retention time analysisand characterization with MALDI-TOF MS at each timepoint Kinetic analysis of target peptide at each time pointwas carried out by least squares analysis of the logarithm ofthe integration peak area versus retention time Nonspecificpeptide degradation was measured by peptide degradationin serum at 0 h Correction for serum peptides that mightcoelute with target peptides was carried out by subtractingintegrated peak areas with equivalent serum only solution atall time points (background subtraction) GraphPad Prism 5was used to analyze the peptide degradation in serum whichwas expressed as the mean ratio plusmn SEM and MicrosoftExcel was used to graph all data Statistical analysis wasconducted using one-way ANOVAwith Newman-Keuls posthoc analysis

3 Results

The selectively S-protected A- and B-chains were of highpurity as determined by analytical RP-HPLC and MALDI-TOF MS An Fmoc-propargylglycine was manually coupledonto the N-terminal end of the B-chain prior to cleavagefrom the solid support and subsequent crude peptide purifi-cation Following this stepwise regioselective disulfide bondformation between the A-chain and monoalkyne molecule-chain was carried out according to established protocols[21ndash23] to form the two-chain three-disulfide bond alkyneH2 relaxin Copper-catalyzed alkyne-azide cycloadditionwasthen utilized to ldquoclickrdquo two molecules of alkyne-H2 relaxinonto a bisazido PEG (PEG)

7 hence forming a dimeric relaxin

molecule separated by a short PEG spacer (Figure 2)Thiswastermed H2 (PEG)

7H2 dimer and was shown to be of high

purity as assessed by analytical RP-HPLC and MALDI-TOFMS (Figure 1)

The CD spectra of H2 (PEG)7H2 were measured in

10mM PBS (pH 74) buffer (Figure 3) The dimer was foundto retain a very similar secondary structure and a high degreeof 120572-helical conformation (with pronounced double minimaat approximately 208 nm and 222 nm) along with some 120573-sheet and random coiled structure The 120572-helical content ofH2 relaxin was found to be 49 helicity compared to theH2-PEG dimer with 48 These values were calculated fromthe MRE at 222 nm the [120579]

222values for relaxin and H2

(PEG)7H2 dimer being minus175114 and minus176794 respectively

The similarities between the MRE and helix content betweenthese two peptides suggested that the dimer essentiallyretained native H2 relaxin-like structure

To assess the biological activity of the dimer binding andactivity in vitro assays were undertaken in comparison to thenative recombinantly producedH2 relaxinThese assays were


NN N

(PEG)7

NH2

O


SH Acm

Acm

B-chain with propargylglycine attached at the N-terminus


StBu

H2N

O

OON3

N37

Regioselective disulfidebond formation

Oxidized A-chain







Alkyne H2 relaxin

ldquoclick reactionrdquo

H2 (PEG)7 H2 dimer

NH

N

N

N

H2N

O

H2N

O

NH

Figure 2 Scheme for the preparation of H2 (PEG)7H2 dimer by solid phase peptide synthesis and click chemistry

carried out in HEK-293T cells stably expressing RXFP1 thenative receptor of H2 relaxin The starting alkyne H2-relaxinwas also assessed for binding and signaling through theRXFP1 and RXFP2 receptor This was performed to confirmthat the disulfide bonds within the twomonomeric analogueswere assembled in the correct form as other combinationsof disulfide pairings have previously been shown to resultin no interaction with the RXFP1 receptor (neither bindingnor activity) [24] Following confirmation of the alkynemonomer possessing full H2 relaxin-like activity (data notshown) the H2 (PEG)

7H2 dimer was tested for binding

and cAMP activity on the same HEK-293T cells expressing

RXFP1 It was found to have similar affinity and potency toH2 relaxin at the RXFP1 receptor (Figures 4(a) and 4(b))Thedimer was then also tested with RXFP2 the native receptorfor INSL3 Interestingly the peptide displayed significantlyweaker activation propensity (Figure 5)

The degradation kinetics and serum stability of thesynthetic H2 (PEG)

7H2 dimer was measured against native

H2 relaxin in male human serum (Figure 6) The purchasedserum was not heat inactivated in order to retain as muchactivity of its proteolytic enzymes and other reductants aspossible This provided a better representation of humanserum and a direct in vitromeasurement in an experimental


H2 relaxinH2 (PEG)7 H2 dimer

200 210 220 230 240 250

Wavelength (nm)

[120579] 222103

deg c

m2

dmol

minus1

10

5

0

minus5

minus10

minus15

minus20

minus25

Figure 3 CD spectroscopic data of the H2 (PEG)7H2 dimer in

comparison with native H2 relaxin

setting of the degradation kinetics of the relaxin dimer whencompared to native relaxin The H2 (PEG)

7H2 dimer was

observed to retain its original dimeric form in vitro with ahalf-life of 252 hrs significantly longer when compared tonative relaxin with 222 hrs

4 Discussion

Nearly 9 decades since its discovery the pleotropic peptideH2 relaxin recently completed a successful Phase III clin-ical trial for the treatment of acute heart failure [13 25]Despite this important success the short in vivo half-lifeof the peptide (10mins) [14 15] necessitates its continuousintravenous infusion for optimum activity As the in vivohalf-life of a peptide or protein is affected by both itsdegradation by enzymes and renal clearance [26 27] it canbe improved by for example conjugation with large MWcompounds such as PEG or by polymerizationaggregationwhich acts to (at least partially) shield it from proteolyticenzymes and also by slowing clearance although best resultsfor the latter occur with a molecular mass of greater than40 kDa [26] Conjugation of PEGmolecules to protein-basedbiopharmaceuticals has recently proved highly successfulin increasing their therapeutic index and clinical use [28]For example a combinational treatment of PEGylated 120572-2-interferon and ribavirin has successfully eradicated hepatitisC virus in approximately 50 of the treated patients leadingto several PEG-based proteins being approved for therapeuticpurposes [29] PEG moieties are also highly hydrated henceincreasing the hydrodynamic radius of the conjugates andcorrespondingly improving solubility and reducing urinaryglomerular filtration rate [30] However such modificationsare not without disadvantagesThe polydispersity of PEG can

100

80

60

40

20

0

Spec

ific b

indi

ng (

)

minus14 minus13 minus12 minus11 minus10 minus9 minus8 minus7 minus6 minus5


Log [peptide] (M)

(a) RXFP1 Eu-H2 binding

100

80

60

40

20

0

Activ

ity (

)



Log [peptide] (M)

(b) RXFP1 cAMP activity

Figure 4 Binding and activity of synthetic H2 (PEG)7H2 dimer in

comparison to native H2 (a) Competition binding of H2 (PEG)7H2

dimer with europium-labeled H2 relaxin in HEK-293T cells stablyexpressing RXFP1 (b) cAMP activity in RXFP1 expressing HEK-293T cells using a pCRE-120573-galactosidase reporter gene system Dataare expressed as percentage of cAMP response and are pooled datafrom at least three independent experiments performed in triplicate

complicate accurate quality control as well as introducingphysicochemical property modifications that make chemicalcharacterization by RP-HPLC andMS extremely challengingdue to peak broadening even disappearance and a lackof ionization For this reason oligomerization strategiesare gaining increasing attention as a means of increasingin vivo stability [31] A dimeric erythropoietin formed bychemical crosslinking of the monomer showed an increasedplasma half-life in rabbits of more than 24 h compared tothe monomerrsquos 4 h The dimer also possessed 26-fold higheractivity in vivo [32] A dimer of the antibacterial peptideA3-APO possessed a substantially increased serum half-life(100min) compared to the monomer (4min) [33] For thisreason we undertook to examine the feasibility of developinga synthetic dimer of H2 relaxin as a first step towards


100

75

50

25

0

cAM

P re

spon

se (

)

minus13 minus12 minus11 minus10 minus9 minus8 minus7 minus6

Log [ligand]

H2 relaxinINSL3PEG-H2 dimer

cAMP stimulation in RXFP2 expressing cells

Figure 5 cAMP activity in RXFP2 expressingHEK-293T cells usinga pCRE-120573-galactosidase reporter gene system Data are expressed aspercentage of cAMP response and are pooled data from at least threeindependent experiments performed in triplicate

obtaining improved pharmacokinetics This was designedand assembled using a bisazido PEG

7moiety between two

molecules of synthetic functionalized H2 relaxin via clickchemistry This linker was chosen because it is sufficientlylonger to space the two peptide units apart and also becauseit is monodisperse thus simplifying characterization Ourprevious studies have shown that the N-terminus of the B-chain can be truncated by six residues [34] or modifiedto accommodate a functional moiety such as a biotin [35]or large fluorophore [18] without significant loss of activityThis is because this site is far from its primary active sitewhich consists of the B-chain C-terminal 120572-helical region[2] Consequently the bisazido PEG

7moiety was conjugated

at both its ends with N-terminal B-chain alkyne H2 relaxinemploying the now well-established click reaction

The CD spectral data showed that H2 (PEG)7H2 dimer

had a comparable secondary structure to native H2 relaxin(Figure 3) strongly suggesting that the dimeric form of H2relaxin has native relaxin-like structural integrity Furtherevidence was also provided by the near-native RXFP1 recep-tor binding and activation activity (Figure 4) This resultconfirmed that the presentation of the active site ofH2 relaxinis essentially unaffected by the tethering of the molecule tothe PEG

7linker via the N-terminus of the B-chain This is

reflected by the in vitro RXFP1 binding and cAMP data inthat despite the dimer being at least twice the molecular sizeof native relaxin its size and spacer length did not impairthe ability of the dimer to bind and activate downstreamRXFP1 signaling The RXFP1 binding domain within the B-chain of the dimer was still exposed and able to interactwith the receptors akin to native relaxin This observationis consistent with previous work where prorelaxin with itsC-chain intact was still able to interact and signal throughthe RXFP1 receptor [36 37] Interestingly and positively

100

80

60

40

20

0

Pept

ide r

emai

ning

()

H2 relaxin

0 1 2 3 4 5 6

Time (hrs)

H2 (PEG)7 H2 dimer

(a)

100

80

60

40

20

0

Pept

ide r

emai

ning

()

0 1 2 3 4 5 6

Time (hrs)

lowastlowast lowast


(b)

Figure 6 In vitro serum stability analysis (a) Curve half-lifedetermination of H2 (PEG)

7H2 dimer when compared to H2

relaxin incubated in vitro at 37∘C in human male serum (b) Bargraph comparison of peptide degradation at different time pointslowast119875 lt 005

at RXFP2 the receptor for the related peptide insulin-likepeptide 3 the dimer was significantly less active than H2relaxin itself (Figure 5)

Importantly in the in vitro serum stability assay theH2 (PEG)

7H2 dimer had an improved increase in its half-

life compared to the native H2 relaxin (Figures 6(a) and6(b)) which is probably due to increasedmolecular shieldingHowever as evidenced by the disparity between the in vitroserum half-life (ca 2 h this study) and reported in vivohalf-life (a few minutes [14 15]) of native H2 relaxin it isclear that renal clearance is a greater issue It remains to bedetermined if the increased hydrodynamic volume due tothe larger molecular weight of the H2 relaxin dimer will alsoresult in a moderation of its renal clearance Past experiencesuggests that this will be the case [33] Indeed it was alsorecently shown that a covalent dimer of exendin-4 had 27


times greater increased biological half-life over the nativemonomer [38]

In conclusion efficient solid phase peptide synthesis incombination with click chemistry techniques was used tosuccessfully prepare a structurally well-defined homodimerof H2 relaxin H2 (PEG)

7H2 This peptide was shown to

possess increased in vitro serum stability compared to nativeH2 relaxin whilst retaining relaxin-like binding affinityactivity and structural integrity in vitro The dimer peptidealso has the potential to slow urinary glomerular filtrationrate [30] which will be investigated in the future Similarapproaches can be adopted for the preparation of furtherincreased multimeric forms of H2 relaxin using for examplea benzene core bearing three or more azido moieties [39] orshort PEG-linked dendrons [31]

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Mohammed Akhter Hossain and John D Wade conceivedand designed the experiments Vinojini B Nair performedthe experiments Ross A D Bathgate and Chrishan S Samuelanalyzed the data Mohammed Akhter Hossain FrancesSeparovic Ross A D Bathgate and John D Wade con-tributed reagentsmaterialsanalysis tools Vinojini B NairMohammed Akhter Hossain Frances Separovic and John DWade wrote the paper Mohammed Akhter Hossain and JohnD Wade equally contributed in the paper

Acknowledgments

This research was partially funded by National Health andMedical Research Council (NHMRC) of Australia ProjectGrants 508995 and 1023078 to John D Wade MohammedAkhter Hossain and Ross A D Bathgate The authorsthank Tania Ferraro and Sharon Layfield for their assistancein undertaking the biological assays Mohammed AkhterHossain was the recipient of a Florey Foundation Fellowshipand Ross A D Bathgate (APP1042650) and Chrishan SSamuel (APP1041766) are NHMRC Senior Research Fellowsand John D Wade (APP5018148) is an NHMRC PrincipalResearch Fellow Research at The Florey Institute of Neu-roscience and Mental Health is supported by the VictorianGovernment Operational Infrastructure Support Program

References

[1] F L Hisaw ldquoExperimental relaxation of the pubic ligamentof the guinea-pigrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 23 pp 611ndash613 1926

[2] M A Hossain and J DWade ldquoThe roles of the A- and B-chainsof human relaxin-2 and -3 on their biological activityrdquo CurrentProtein and Peptide Science vol 11 no 8 pp 719ndash724 2010

[3] R A D Bathgate M L Halls E T van der Westhuizen GE Callander M Kocan and R J Summers ldquoRelaxin family

peptides and their receptorsrdquo Physiological Reviews vol 93 no1 pp 405ndash480 2013

[4] G D Bryant-Greenwood ldquoRelaxin as a new hormonerdquoEndocrine Reviews vol 3 no 1 pp 62ndash90 1982

[5] R A Bathgate R Ivell B M Sanborn O D Sherwood andR J Summers ldquoInternational union of pharmacology LVIIrecommendations for the nomenclature of receptors for relaxinfamily peptidesrdquo Pharmacological Reviews vol 58 no 1 pp 7ndash31 2006

[6] X-J Du R A D Bathgate C S Samuel A M Dart and R JSummers ldquoCardiovascular effects of relaxin from basic scienceto clinical therapyrdquo Nature Reviews Cardiology vol 7 no 1 pp48ndash58 2010

[7] F Shabanpoor F Separovic and J D Wade ldquoThe humaninsulin superfamily of polypeptide hormonesrdquo Vitamins andHormones vol 80 pp 1ndash31 2009

[8] C Eigenbrot M Randal C Quan J Burnier L OrsquoConnell andE A A Rinderknecht Kossiakoff ldquoX-ray structure of humanrelaxin at 15 Ardquo Journal of Molecular Biology vol 221 no 1 pp15ndash21 1991

[9] S J Shire L AHolladay and E Rinderknecht ldquoSelf-associationof human and porcine relaxin as assessed by analytical ultracen-trifugation and circular dichroismrdquo Biochemistry vol 30 no 31pp 7703ndash7711 1991

[10] K Huus S Havelund H B Olsen M Van De Weert andS Frokjaer ldquoThermal dissociation and unfolding of insulinrdquoBiochemistry vol 44 no 33 pp 11171ndash11177 2005

[11] J Brange and L Langkjoer ldquoInsulin structure and stabilityrdquoPharmaceutical biotechnology vol 5 pp 315ndash350 1993

[12] T N Vinther M Norrman H M Strauss et al ldquoNovelcovalently linked insulin dimer engineered to investigate thefunction of insulin dimerizationrdquo PLoS ONE vol 7 no 2Article ID e30882 2012

[13] J R Teerlink G Cotter B A Davison et al ldquoSerelaxinrecombinant human relaxin-2 for treatment of acute heartfailure (RELAX-AHF) a randomised placebo-controlled trialrdquoThe Lancet vol 381 no 9860 pp 29ndash39 2013

[14] SAChenA J PerlmanN Spanski et al ldquoThepharmacokinet-ics of recombinant human relaxin in nonpregnant women afterintravenous intravaginal and intracervical administrationrdquoPharmaceutical Research vol 10 no 6 pp 834ndash838 1993

[15] S A Chen B Reed T Nguyen N Gaylord G B Fullerand J Mordenti ldquoThe pharmacokinetics and absorption ofrecombinant human relaxin in nonpregnant rabbits and rhesusmonkeys after intravenous and intravaginal administrationrdquoPharmaceutical Research vol 10 no 2 pp 223ndash227 1993

[16] C S Samuel F Lin M A Hossain et al ldquoImproved chemicalsynthesis and demonstration of the relaxin receptor bindingaffinity and biological activity of mouse relaxinrdquo Biochemistryvol 46 no 18 pp 5374ndash5381 2007

[17] F Shabanpoor R A D Bathgate A Belgi et al ldquoSite-specificconjugation of a lanthanide chelator and its effects on thechemical synthesis and receptor binding affinity of humanrelaxin-2 hormonerdquoBiochemical and Biophysical Research Com-munications vol 420 no 2 pp 253ndash256 2012

[18] L J Chan C M Smith B E Chua et al ldquoSynthesis of fluores-cent analogs of relaxin family peptides and their preliminary invitro and in vivo characterizationrdquo Frontiers in Chemistry vol1 article 30 2013

[19] L V Najbar D J Craik J D Wade D Salvatore and MJ McLeish ldquoConformational analysis of LYS(11-36) a peptide


derived from the 120573- sheet region of T4 lysozyme in TFE andSDSrdquo Biochemistry vol 36 no 38 pp 11525ndash11533 1997

[20] P G Scott C M Dodd E M Bergmann J K Sheehan and PN Bishop ldquoCrystal structure of the biglycan dimer and evidencethat dimerization is essential for folding and stability of classI small leucine-rich repeat proteoglycansrdquo Journal of BiologicalChemistry vol 281 no 19 pp 13324ndash13332 2006

[21] M A Hossain A Belgi F Lin et al ldquoUse of a temporaryldquosolubilizingrdquo peptide tag for the Fmoc solid-phase synthesis ofhuman insulin glargine via use of regioselective disulfide bondformationrdquoBioconjugate Chemistry vol 20 no 7 pp 1390ndash13962009

[22] M A Hossain R A D Bathgate C K Kong et al ldquoSynthesisconformation and activity of human insulin-like peptide 5(INSL5)rdquo ChemBioChem vol 9 no 11 pp 1816ndash1822 2008

[23] M A Hossain K J Rosengren S Zhang et al ldquoSolid phasesynthesis and structural analysis of novel A-chain dicarbaanalogs of human relaxin-3 (INSL7) that exhibit full biologicalactivityrdquo Organic and Biomolecular Chemistry vol 7 no 8 pp1547ndash1553 2009

[24] M P Del Borgo R A Hughes and J DWade ldquoConformation-ally constrained single-chain peptide mimics of relaxin B-chainsecondary structurerdquo Journal of Peptide Science vol 11 no 9 pp564ndash571 2005

[25] J R Teerlink M Metra G M Felker et al ldquoRelaxin for thetreatment of patients with acute heart failure (Pre-RELAX-AHF) a multicentre randomised placebo-controlled parallel-group dose-finding phase IIb studyrdquo The Lancet vol 373 no9673 pp 1429ndash1439 2009

[26] M Werle and A Bernkop-Schnurch ldquoStrategies to improveplasma half life time of peptide and protein drugsrdquoAminoAcidsvol 30 no 4 pp 351ndash367 2006

[27] L Otvos and J D Wade ldquoCurrent challenges in peptide-baseddrug discoveryrdquo Frontiers in Chemistry vol 2 article 62 2014

[28] M W Fried M L Shiffman K Rajender Reddy et alldquoPeginterferon alfa-2a plus ribavirin for chronic hepatitis Cvirus infectionrdquoThe New England Journal of Medicine vol 347no 13 pp 975ndash982 2002

[29] J M Harris and R B Chess ldquoEffect of pegylation on pharma-ceuticalsrdquoNature Reviews Drug Discovery vol 2 no 3 pp 214ndash221 2003

[30] P Caliceti and F M Veronese ldquoPharmacokinetic and biodistri-bution properties of poly(ethylene glycol)-protein conjugatesrdquoAdvanced Drug Delivery Reviews vol 55 no 10 pp 1261ndash12772003

[31] E R Gillies and J M J Frechet ldquoDendrimers and dendriticpolymers in drug deliveryrdquo Drug Discovery Today vol 10 no1 pp 35ndash43 2005

[32] A J Sytkowski E D Lunn K L Davis L Feldman andS Siekman ldquoHuman erythropoietin dimers with markedlyenhanced in vivo activityrdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 95 no 3 pp 1184ndash1188 1998

[33] P B Noto G Abbadessa M Cassone et al ldquoAlternativestabilities of a proline-rich antibacterial peptide in vitro and invivordquo Protein Science vol 17 no 7 pp 1249ndash1255 2008

[34] MAHossain K J Rosengren C S Samuel et al ldquoTheminimalactive structure of human relaxin-2rdquo Journal of BiologicalChemistry vol 286 no 43 pp 37555ndash37565 2011

[35] M N Mathieu J D Wade G W Tregear et al ldquoSynthesisconformational studies and biological activity of N120572-mono-biotinylated rat relaxinrdquo Journal of Peptide Research vol 57 no5 pp 374ndash382 2001

[36] R A Bathgate A Hsueh and O D Sherwood ldquoPhysiology andmolecular biology of the relaxin peptide familyrdquo in Knobil andNeillrsquos Physiology of Reproduction J D Neill Ed pp 679ndash768Academic Press San Diego Calif USA 2006

[37] M A Hossain C S Samuel C Binder et al ldquoThe chemicallysynthesized human relaxin-2 analog B-R1317K H2 is anRXFP1 antagonistrdquo Amino Acids vol 39 no 2 pp 409ndash4162010

[38] T H Kim H H Jiang S Lee et al ldquoMono-PEGylated dimericexendin-4 as high receptor binding and long-acting conjugatesfor type 2 anti-diabetes therapeuticsrdquo Bioconjugate Chemistryvol 22 no 4 pp 625ndash632 2011

[39] C Heinis T Rutherford S Freund and G Winter ldquoPhage-encoded combinatorial chemical libraries based on bicyclicpeptidesrdquo Nature Chemical Biology vol 5 no 7 pp 502ndash5072009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


insulin is involved in its tyrosine kinase receptor activationwhereas its dimeric and hexameric forms are involved instabilizing the molecule during storage [10 11] A covalentlylinked (through a disulfide bridge) dimeric insulin peptidewas recently prepared by recombinant DNA technology Itneither bound to the insulin receptor nor induced ametabolicresponse in vitro [12] which is consistent with the view thatthe monomeric form is the active form of insulin Howeverthe insulin dimer was shown to be extremely thermodynam-ically stable in vitro which highlighted the importance ofoligomerization for insulin stability [12] This suggests thata covalently linked dimeric relaxin may also be more stablein vitro as well as in vivo compared to its monomeric formSuch a compound if it retained biological activity would bevery valuable given that H2 relaxin recently passed PhaseIII clinical trials for treating acute heart failure [13] despitehaving a short in vivo half-life of approximately 10min [14 15]that is characteristic of many peptides and proteins It is forthis reason that H2 relaxin requires continuous intravenousinfusion into patients over 48 hours [13] Therefore there isa clear need for improving the pharmacokinetic propertiesof H2 relaxin in order to potentially improve its therapeuticvalue

In this study we undertook to design and develop a cova-lently linked dimeric analogue of synthetic H2 relaxin usingboth click chemistry and a small polyethylene glycol (PEG)spacer to link the two monomers in such a structural orien-tation so as to retain biological activity (Figure 1) We studiedits structural and functional role using circular dichroism(CD) spectroscopy and RXFP1- and RXFP2-expressing cellsrespectively It was shown that the dimeric H2 relaxinpossessed a high degree of secondary structural similarityto native H2 relaxin Importantly unlike the insulin dimerthe dimeric H2 relaxin is equipotent to native H2 relaxinmonomer and exhibits improved in vitro serum stability

2 Experimental Procedures

21 Materials 9-Fluorenylmethoxycarbonyl- (Fmoc-) pro-tected L-120572-amino acids and 1-[bis(dimethylamino)methy-lene]-1H-benzotriazolium hexafluorophosphate 3-oxide(HBTU) were purchased from GL Biochem (ShanghaiChina) NN-Dimethylformamide (DMF) piperidine andtrifluoroacetic acid (TFA) were obtained from Auspep(Melbourne Australia) PAL-PEG-PS resins with substitu-tion of ca 020mmolg were purchased from AppliedBiosystems Inc (Melbourne Australia) Diethyl ethermethanol acetonitrile and dichloromethane were purchasedfrom Merck (Melbourne Australia) trifluoromethanes-ulfonic acid (TFMSA) anisole triisopropylsilane (TIPS) 36-dioxa-18-octanedithiol (DODT) and diisopropylethylamine(DIEA) were from Sigma-Aldrich (Sydney Australia)and 221015840-dipyridyl disulfide (DPDS) was from Fluka(Switzerland) CaCl

2was purchased from Mallinckrodt

(Melbourne Australia) and Na2HPO4and MgSO

4were pur-

chased from BDH Chemicals while KH2PO4

andMnCl

2were purchased from Ajax Chemicals (Sydney

Australia) Dulbeccorsquos modified Eagle medium (DMEM)penicillinstreptomycin L-glutamine and fetal bovine serum

were purchased from Invitrogen (Melbourne Australia)Sterile-filtered serum from human AB plasma was purchasedfrom Sigma-Aldrich (Sydney Australia) All other reagentswere purchased from Sigma-Aldrich (Sydney Australia)Native recombinant DNA-derived human relaxin-2 used ascontrol was obtained from Corthera Inc (San Francisco CAa subsidiary of Novartis AG Basel Switzerland) Syntheticeuropium-labelled H2 relaxin unlabelled H2 and humanINSL3 were prepared in-house as previously described[16 17]

22 Synthesis

221 Chemical Peptide Synthesis The H2 relaxin A- and B-chains were assembled as C-terminal amides on an auto-mated Protein Technologies Tribute peptide synthesizer(Tuscan AZ) or CEM Liberty microwave peptide synthesizer(Ai Scientific Australia) using Fmoc chemistry Side chainprotecting groups of trifunctional amino acids were TFA-labile except for tert-butyl- (tBu-) protected cysteine inposition A11 and acetamidomethyl- (Acm-) protected cys-teines in positions A24 and B23 Using instrument defaultprotocols both A- and B-chains were separately synthesizedeither at 01mmol scale or 02mmol scale activated witheither 4- or 5-fold molar excess of HBTU (04 or 05mmolfor a 01mmol scale of resin 08mmol for a 02mmol scaleof resin) in the presence of 5 equivalents DIEA Resin-attached peptides were treated with 20 vv piperidineDMFto remove N120572-Fmoc protecting groups When using themicrowave synthesizer coupling and deprotection steps werecarried out at 75∘C and 25W microwave power for 5minsand 60W microwave power for 3mins respectively whenusing the CEM Liberty microwave synthesizer Couplingand deprotection steps were carried out for 30 and 10minsrespectively when using Tribute synthesizers Upon completecoupling of the final amino acid of the native B-chain peptidesequence an extra amino acid containing the alkyne group(Fmoc-L-propargylglycine) was coupled by manual couplingprocedures

222 Peptide-Resin Cleavage Upon completion of solidphase synthesis the A- and B-chains were cleaved fromthe solid supports by treatment with TFA containinganisoleTIPSDODT (9425215 20mL) for 2 hCleaved products were concentrated byN

2bubbling precipi-

tated with ice-cold diethyl ether and centrifuged at 3000 rpmfor 5mins The centrifuged pellet was then washed with ice-cold diethyl ether and centrifugedThis process was repeatedat least three times

223 Peptide Purification All RP-HPLC analytical andpurification reactions were monitored using analytical andpreparativeVydac C18 columns (pore size 300 A particle size46times 250mmor 22times 250mm resp) in a gradientmodewitheluant A 01 aq TFA and eluant B 01TFA in acetonitrile

224 Preparation of Monoalkyne H2 Relaxin Followingsimultaneous cleavage and side chain deprotection and






NN N

N

N

N

(PEG)7

NH2

H2N

O

O

(a)

0 10 20 30

1520

Time (min)

(b)

6000 10000 14000 18000

1255362

mz

(c)


125300



7bisazide


7











3 Results













NN N

(PEG)7

NH2

O


SH Acm

Acm



StBu

H2N

O

OON3

N37


Oxidized A-chain







Alkyne H2 relaxin


H2 (PEG)7 H2 dimer

NH

N

N

N

H2N

O

H2N

O

NH











200 210 220 230 240 250

Wavelength (nm)

[120579] 222103

deg c

m2

dmol

minus1

10

5

0

minus5

minus10

minus15

minus20

minus25




7H2 dimer was


4 Discussion


100

80

60

40

20

0

Spec

ific b

indi

ng (

)



Log [peptide] (M)


100

80

60

40

20

0

Activ

ity (

)



Log [peptide] (M)







100

75

50

25

0

cAM

P re

spon

se (

)


Log [ligand]





7moiety between two








100

80

60

40

20

0

Pept

ide r

emai

ning

()

H2 relaxin

0 1 2 3 4 5 6

Time (hrs)

H2 (PEG)7 H2 dimer

(a)

100

80

60

40

20

0

Pept

ide r

emai

ning

()

0 1 2 3 4 5 6

Time (hrs)

lowastlowast lowast


(b)

















Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






NN N

N

N

N

(PEG)7

NH2

H2N

O

O

(a)

0 10 20 30

1520

Time (min)

(b)

6000 10000 14000 18000

1255362

mz

(c)


125300



7bisazide


7











3 Results













NN N

(PEG)7

NH2

O


SH Acm

Acm



StBu

H2N

O

OON3

N37


Oxidized A-chain







Alkyne H2 relaxin


H2 (PEG)7 H2 dimer

NH

N

N

N

H2N

O

H2N

O

NH











200 210 220 230 240 250

Wavelength (nm)

[120579] 222103

deg c

m2

dmol

minus1

10

5

0

minus5

minus10

minus15

minus20

minus25




7H2 dimer was


4 Discussion


100

80

60

40

20

0

Spec

ific b

indi

ng (

)



Log [peptide] (M)


100

80

60

40

20

0

Activ

ity (

)



Log [peptide] (M)







100

75

50

25

0

cAM

P re

spon

se (

)


Log [ligand]





7moiety between two








100

80

60

40

20

0

Pept

ide r

emai

ning

()

H2 relaxin

0 1 2 3 4 5 6

Time (hrs)

H2 (PEG)7 H2 dimer

(a)

100

80

60

40

20

0

Pept

ide r

emai

ning

()

0 1 2 3 4 5 6

Time (hrs)

lowastlowast lowast


(b)

















Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






3 Results













NN N

(PEG)7

NH2

O


SH Acm

Acm



StBu

H2N

O

OON3

N37


Oxidized A-chain







Alkyne H2 relaxin


H2 (PEG)7 H2 dimer

NH

N

N

N

H2N

O

H2N

O

NH











200 210 220 230 240 250

Wavelength (nm)

[120579] 222103

deg c

m2

dmol

minus1

10

5

0

minus5

minus10

minus15

minus20

minus25




7H2 dimer was


4 Discussion


100

80

60

40

20

0

Spec

ific b

indi

ng (

)



Log [peptide] (M)


100

80

60

40

20

0

Activ

ity (

)



Log [peptide] (M)







100

75

50

25

0

cAM

P re

spon

se (

)


Log [ligand]





7moiety between two








100

80

60

40

20

0

Pept

ide r

emai

ning

()

H2 relaxin

0 1 2 3 4 5 6

Time (hrs)

H2 (PEG)7 H2 dimer

(a)

100

80

60

40

20

0

Pept

ide r

emai

ning

()

0 1 2 3 4 5 6

Time (hrs)

lowastlowast lowast


(b)

















Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


NN N

(PEG)7

NH2

O


SH Acm

Acm



StBu

H2N

O

OON3

N37


Oxidized A-chain







Alkyne H2 relaxin


H2 (PEG)7 H2 dimer

NH

N

N

N

H2N

O

H2N

O

NH











200 210 220 230 240 250

Wavelength (nm)

[120579] 222103

deg c

m2

dmol

minus1

10

5

0

minus5

minus10

minus15

minus20

minus25




7H2 dimer was


4 Discussion


100

80

60

40

20

0

Spec

ific b

indi

ng (

)



Log [peptide] (M)


100

80

60

40

20

0

Activ

ity (

)



Log [peptide] (M)







100

75

50

25

0

cAM

P re

spon

se (

)


Log [ligand]





7moiety between two








100

80

60

40

20

0

Pept

ide r

emai

ning

()

H2 relaxin

0 1 2 3 4 5 6

Time (hrs)

H2 (PEG)7 H2 dimer

(a)

100

80

60

40

20

0

Pept

ide r

emai

ning

()

0 1 2 3 4 5 6

Time (hrs)

lowastlowast lowast


(b)

















Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



200 210 220 230 240 250

Wavelength (nm)

[120579] 222103

deg c

m2

dmol

minus1

10

5

0

minus5

minus10

minus15

minus20

minus25




7H2 dimer was


4 Discussion


100

80

60

40

20

0

Spec

ific b

indi

ng (

)



Log [peptide] (M)


100

80

60

40

20

0

Activ

ity (

)



Log [peptide] (M)







100

75

50

25

0

cAM

P re

spon

se (

)


Log [ligand]





7moiety between two








100

80

60

40

20

0

Pept

ide r

emai

ning

()

H2 relaxin

0 1 2 3 4 5 6

Time (hrs)

H2 (PEG)7 H2 dimer

(a)

100

80

60

40

20

0

Pept

ide r

emai

ning

()

0 1 2 3 4 5 6

Time (hrs)

lowastlowast lowast


(b)

















Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100

75

50

25

0

cAM

P re

spon

se (

)


Log [ligand]





7moiety between two








100

80

60

40

20

0

Pept

ide r

emai

ning

()

H2 relaxin

0 1 2 3 4 5 6

Time (hrs)

H2 (PEG)7 H2 dimer

(a)

100

80

60

40

20

0

Pept

ide r

emai

ning

()

0 1 2 3 4 5 6

Time (hrs)

lowastlowast lowast


(b)

















Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article synthetic covalently linked dimeric form

Documents