growth hormone stability paper

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The Interaction of Heparin/Polyanions with Bovine,Porcine, and Human Growth Hormone

SANGEETA B. JOSHI, TIM J. KAMERZELL, CHRIS McNOWN, C. RUSSELL MIDDAUGH

Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047

Received 11 February 2007; revised 22 April 2007; accepted 1 May 2007

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21056

ABSTRACT: The interaction of polyanions with proteins is of potential pharmaceuticaland cellular significance. A partial thermodynamic description of the interaction of fourrepresentative polyanions with human, bovine, and porcine growth hormone isdescribed. A heparin bead-binding assay confirms all growth hormones bind to heparin

but to varying extents. Moderate-binding constants and high ratios of bound protein tothe more extended polyanions, heparin, and dextran sulfate were measured by iso-thermal titration calorimetry and dynamic light scattering. The binding constants andratio of protein bound to ligand were significantly smaller for the low molecular weightpolyanions phytic acid and sucrose octasulfate (SOS). The effect of polyanion binding onthe bovine, porcine, and human growth hormones (hGH) structural and colloidal

stability was also explored. Heparin and dextran sulfate inhibit porcine somatotropin(pST) and bovine somatotropin (bST) aggregation to the greatest extent, as compared tophytic acid and SOS, while decreasing secondary and tertiary structural stability asmeasured by the temperature dependence of their circular dichroism and intrinsic

fluorescence. Somewhat surprisingly, the polyanions do not appear to affect the struc-ture or stability of hGH. The potential biological significance of growth hormone

polyanion interactions is discussed. 2007 Wiley-Liss, Inc. and the American PharmacistsAssociation J Pharm Sci

Keywords: thermodynamics; polyanion; heparin; fluorescence; circular dichroism;isothermal titration calorimetry; stability; growth hormone

INTRODUCTION

Growth hormones are globular proteins of$191amino acids.1 Also known as the pituitarysomatotropins, they are involved in a wide varietyof biological functions including growth and

metabolism. The structure of bovine (bST, bovine

somatotropin), porcine (pST, porcine somatotro-pin), and human growth hormone (hGH) aresimilar with greater than 90% sequence homologybetween bST and pST.2 The crystal structure hasbeen solved for a modified Met-pST analogue,3

and for affinity matured,4 and apo wild-type hGH

at 2.5 A resolution.5

The structure shows that thegrowth hormones consist of a four alpha helixbundle motif with a left-handed super-helicaltwist.

All three growth hormones contain lysine, his-tidine, and arginine rich clusters suggestive ofsites for potential electrostatic interactions withnegatively charged ligands. In fact, hGH has beenshown to interact with the polyanion heparinleading to its stabilization against interfacialunfolding.6 Electrostatic interactions between

SangeetaB. Joshi and TimJ. Kamerzell contributed equallyto this work.

Abbreviations: pST, porcine somatotropin; bST, bovinesomatotropin; hGH, human growth hormone; DS, dextransulfate; PA, phytic acid; REES, red edge excitation spectro-scopy; ITC, isothermal titration calorimetry; CD, circulardichroism; DLS, dynamic light scattering.

Correspondence to: C. Russell Middaugh (Telephone: 785-864-5813; Fax: 785-864-5814; E-mail: [email protected])

Journal of Pharmaceutical Sciences

2007 Wiley-Liss, Inc. and the American Pharmacists Association

JOURNAL OF PHARMACEUTICAL SCIENCES 1


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positively charged protein residues and variouspolyanionic compounds have been shown to sig-nificantly enhance the physical stability andactivity of some protein molecules,714 many ofthem hormones. For example, human FGF1 isunstable at physiological temperatures but its

physical stability is markedly increased uponaddition of a wide variety of polyanions.13,14

Furthermore, RNA and heparin both bind tooverlapping sites of epidermal growth factor likedomains of Factor VII activating protease andincrease activity15 in similar fashion as theactivation of Factor XII by heparin and chon-droiton sulfate E.16

It is now well established that in the case ofseveral growth factors, the interaction of theproteins with cell surface proteoglycans is ofphysiological significance. It has been proposed

and experimental data has been obtained insupport of the idea that some growth factorsmay be presented bound to molecules such asheparan and chondroitan sulfate to their protei-naceous cell surface receptors. This may facilitatethe oligomerization of target receptors initiatingfurther signaling events. It has also been postu-lated that the proteoglycan may serve as storagesites for such hormones with their release byheparinase and related enzymes regulating theirinteraction with receptors. There is little evi-dence, however, that the growth hormones areinvolved in any such activities. In this work, avariety of methods are employed to explore thepotential interaction of polyanions with human,bovine, and porcine growth hormones. The poly-anions, dextran sulfate ($50000 amu), heparin($1216000 amu), phytic acid ($923 amu), andsucrose octasulfate (SOS; $1287 amu) were used.Heparin was chosen for its similarity to the cellsurface proteoglycans, while the other polyanionswere investigated because of their ranges ofsize and charge density which can be used toprobe the effect of varying polyanionicity. Theeffect of polyanion binding on all three growth

hormones, including a partial thermodynamicdescription, a determination of the number ofproteins bound per ligand, associated secondaryand tertiary structural changes, and the effect oninhibition of protein aggregation are described.Surprisingly, we find that bovine and porcinegrowth hormone bind a variety of polyanions withmoderate binding constants but with a reductionof their thermal stabilization, while hGH exhibitsmuch less interaction with all of the polyanionsstudied.

EXPERIMENTAL PROCEDURES (MATERIALSAND METHODS)

Materials

Bovine growth hormone (bST) was obtained from

Monsanto (St. Louis, MO). Porcine growth hor-mone (pST) from Monsanto and Pfizer, Inc., andhGH from Pharmacia, Inc. (Piscataway, NJ). Allthree proteins produce a single band upon SDSPAGE. Dextran sulfate was purchased fromSpectrum (New Brunswick, NJ), heparin andphytic acid from Sigma (St. Louis, MO) and SOSfrom TRC, Inc. (Ontario, Canada). All other chemi-cals were of reagent grade and were obtained fromSigma and Fisher Scientific (Pittsburgh, PA).

Methods

Protein Sample Preparation

Bovine and porcine growth hormones wereobtained as lyophilized solids and were stored at208C. hGH was obtained as a concentratedprotein solution in Hepes buffer and was stored at208C. bST protein solutions were prepared bydissolving bST (1 mg/mL) in bicarbonate buffer(35 mM, pH 9.5) followed by overnight dialysisagainst Hepes buffer (20 mM Hepes, pH 8.0).The dialyzed protein solution was centrifuged for5 min at 14000 rpm to remove aggregated protein

before concentration determination. Protein con-centration was determined at room temperatureby absorbance measurement at 280 nm (A280 nm0.65 at 1 cm, 0.1%) using an Agilent 8453 UV-

Visible spectrophotometer (Palo Alto, CA) fittedwith a Peltier temperature controller. The samplesolutions of pST and hGH were prepared bydiluting the concentrated stock solutions ofproteins in 20 mM Hepes buffer, pH 8.0.

Heparin Bead-Binding Isotherms

Samples were prepared in 20 mM Hepes, pH 8.0

by adding increasing amounts of protein (bST,pST, hGH) to a solution containing 100 uL ofheparinagarose beads (Sigma, St. Louis, MO) in2 mL polypropylene microcentrifuge tubes. Thetotal sample volume was 200 mL. The sampleswere placed on a rotating mixer for 1 h at 48C andthen centrifuged at 14000 rpm for 2 min in amicrocentrifuge. The unbound protein in thesupernatant was measured by the UV absorbanceat 280 nm and was subtracted from the totalprotein to calculate the amount of heparin bound

JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps

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protein. Protein concentration was determined byemploying the extinction coefficients of 0.65, 0.66,and 0.85 (A280 nm at 1 mg/mL) for bST, pST, andhGH, respectively. The samples of pST in thepresence of heparin beads were also prepared inHepes buffer containing either 0.15 or 1.0 M NaCl.

Isothermal Titration Calorimetry (ITC)

All calorimetric titrations were performed using aCSC Model 4200 isothermal titration calorimeter(Calorimetry Sciences, Lindon, UT) at 258C andwere performed with full sample (1300 mL) andreference cells, with the sample cell stirrer set at297 rpm. The reference cell contained purifiedwater and all solutions were degassed before use.

ITCRun Software (Calorimetry Science) wasused for instrument operation and collection of

data. The differential heats of binding wereintegrated and analyzed utilizing Bindworks3.0 software. Data collected from all experimentswere fit to an independent set of multiple bindingsites model with the total heat determined by:

Q VDH

L1 MnK

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1 MnK LK2 4KL

p2K

" #

(1)

where DH is the enthalpy of binding, K is thebinding constant, n is the number of binding sites,V is the volume of the cell, [L] the total ligandconcentration, and [M] the protein concentra-tion.17 Titrations consisted of 3001200-s equili-bration times with 5-min intervals betweeninjections. Titrations of polyanion into macromo-lecule utilized a titration regimen of 25 10 mL or405 mL. Heats of dilution were accounted for bysubtracting the integrated heats of dilution fromthe binding heats.

Dynamic Light Scattering (DLS)

Dynamic light scattering measurements wereperformed with a Brookhaven Model BI200SMinstrument (Brookhaven, NY). Light scatteringwas detected at 908 using a continuous wave 532 nmdiode pumped solid-state laser. All samples werefiltered prior to titration. DLS measurementswere conducted under conditions which simulatedthe ITC experiments. Ten microliters of ligandwere directly injected into the macromolecular

solution, stirred, and equilibrated for $1200 sbefore measurements were taken. Five runs ateach ratio of macromolecule to ligand wereperformed, and repeated three times. Particlesize distributions were analyzed by multiplemethods. Titrations were initially analyzed by

the method of cumulants using a gaussiananalysis, least squares fit. For more complexmultimodal distributions, data were analyzedemploying a nonlinear, least squares calculation(with a nonnegative constraint) of the inverseLaplace transform of the autocorrelation function.Furthermore, all calculations were performedusing both a number and intensity average ofthe population. The resulting diffusion coeffi-cients were converted to hydrodynamic radiiusing the Stokes/Einstein equation.

Red Edge Excitation Spectroscopy (REES)Red edge excitation fluorescence (REE) measure-ments were conducted with a JASCO FP-6500spectrofluorometer (Tokyo, Japan). Fluorescenceemission spectra were collected after varying theexcitation wavelength every 1 nm from 285310 nm. Collection of emission spectral data began7 nm past the excitation wavelength with thetemperature held at 108C. A bandwidth of 3 nmwas used for both emission and excitation. Theligands, dextran sulfate and heparin were com-bined with each growth hormone in a 1:1 molar

ratio, while SOS and phytic acid were used at a 1:1weight ratio ($17:1 and 24:1 molar ratio SOS:GHand PA:GH). Emission maxima were determinedutilizing 1st and 2nd derivative analysis. A centerof mass determination was also used to find theemission lmax. For spectral center of massdetermination, Eq. (2) was used

C:M:

Plem FiP

Fi(2)

where lem is the emission wavelength, and Fi isthe fluorescence intensity at the respective emis-

sion wavelength.

Turbidity Studies

The kinetics of protein aggregation was monitoredby increases in the turbidity at 360 nm using aFluostar Galaxy microplate reader (BMG Lab-technologies, Offenberg, Germany). Preliminarystudies established solution conditions underwhich the growth hormones exhibit convenientlymeasurable aggregation behavior. The aggrega-tion of proteins (bST and pST, 0.02 mM) in Hepes

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES

GROWTH HORMONES ARE HEPARIN-BINDING PROTEINS 3


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buffer (20 mM Hepes, pH 8.0) was monitoredcontinuously at 378C in the presence of DTT (finalconcentration 30 mM) for 2 h. Aggregation studiesof protein samples were performed both with andwithout polyanions with the aggregation seenwith protein alone used as a control. The max-

imum OD observed in control samples at 2 h wasused as the maximum extent of aggregation sinceaggregation was complete by this time. Inhibitionof aggregation by polyanions was then character-ized by their ability to lower the maximum ODobserved after 2 h.

Circular Dichroism Studies

CD studies were performed with a Jasco 720spectrophotometer (Tokyo, Japan) equipped witha Peltier temperature controller. Thermal unfold-ing experiments were performed at 0.18C inter-vals using a 158C/h temperature ramp rate tomonitor the change in ellipticity at 222 nm as afunction of temperature. A protein concentrationof 7.0 mM was used in all the studies. Samples ofGH containing various polyanions, a 1:1 molarratio of protein to dextran sulfate and heparin anda 1:1 weight ratio of protein to phytic acid and SOS($17:1 and 24:1 molar ratio SOS:GH and PA:GH)were contained in a 0.1-cm path length cell sealedwith a Teflon stopper. A resolution of 0.1 nm and ascanning speed of 20 nm/min with a 2-s responsetime were employed. The CD signals were

converted to molar ellipticity using the JascoSpectra Manager software.

Fluorescence Spectroscopy

Fluorescence studies were performed using a PTIQuanta Master Spectrophotometer (Lawrence-ville, NJ) equipped with a thermostated cuvetteholder. A protein concentration of 9.0 mM wasused in all studies and the amount of polyanionwas the same as described above. The intrinsicfluorescence spectrum of tryptophan was mon-itored using an excitation wavelength of 295 nm

(>95% Trp emission). Emission spectra werecollected over a range of 305435 nm. Excitationand emission slits were set at 4 nm and a 1-cmpath length quartz cuvette was used in allexperiments. The spectra were collected from108C to 858C at 2.58C intervals with a 5-minequilibration time at each temperature. Bufferbaselines were subtracted from each spectrumprior to data analysis. Data analysis was per-formed using FelixTM (PTI) software. Emissionpeak positions were determined by first derivative

analysis. Reproducibility of the thermal unfoldingtemperature (Tm, the midpoint of the transition)was 28C.

RESULTS

Binding to Heparin-Conjugated Agarose Beads

Initially, the binding of human, bovine, andporcine growth hormones to heparinized beadswas confirmed by a simple binding assay. Eachgrowth hormone in increasing amounts wasincubated with heparin-derivatized agarose beadsand the extent of adsorption was determined bythe measurement of protein remaining in solutionemploying UV absorbance at 280 nm. All growthhormones bound heparin beads but to varyingextents (Fig. 1). The amount of protein boundrelative to the amount added was greatest for bST,with pST having intermediate values, and hGHbinding the least. The binding assay was alsoconducted in the presence of various concentra-tions of sodium chloride. The extent of binding ofprotein with heparin beads decreased withincreasing salt concentration (Fig. 1), suggestingthe expected involvement of electrostatic interac-tions in the binding process.

ITC Titrations

A thermodynamic description of the growth

hormones interactions with various polyanionsis shown in Table 1. A representative thermogram

Figure 1. Binding isotherms of bovine, porcine, andhuman growth hormones to heparin beads in 20 mMhepes buffer, pH 8.0 at various ionic strengths. (&) bST

without salt, (~) hGH without salt, (&) pST withoutsalt, (~) pST with 0.15M NaCl, (*) and pST with 1.0 MNaCl. Each point represents an average of threeexperiments.


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of pST (3.71 mg/mL) titrated with dextran sulfate(2.2 mg/mL) at 258C using a program of 25injections of 10mL is shown in Figure 2. The heatsof dilution were calculated by a blank titration ofligand into dialysis buffer and subtracted in thefinal analysis. The integrated peak areas were fit

to an independent set of multiple binding sitesmodel from which the parametersDH(enthalpy ofbinding in kJ/mol ligand), K (binding constant),and n (binding stoichiometry) were obtained.Utilizing the equation DGRTlnK, the Gibbsfree energy was calculated and subsequentlythe entropy from DGDHTDS. Because ofthe large number of binding sites demonstratedfor dextran sulfate and heparin, the apparententhalpy of interaction was divided by the total

Table

1.

ThermodynamicAnalysisofGrowthHormone/PolyanionInteractions

Protein

Ligand

DH

(kJ/mol)

Adj.DH

(kJ/Charge

)

DG(kJ/mol)

DS(J/molK)

K(M

1)

n

ITCProt/Sacch

DLSProt/Sacch

pST

Dextransulfate

1080

20

3.4

37

3500

2.8

E6

2.7

E5

0.0

2

1/3

$1/3

pST

Heparin

436

21

7.4

33

1350

5.1

E5

2.5

E4

0.0

3

1/1.3

$1/1.5

pST

Sucroseoctasulfate

82

20

10.3

25

190

2.3

E4

4.4

E3

0.1

NA

NA

pST

Phyticacid

7

3

1.2

35

94

4.3

E5

2.5

E5

0.3

NA

NA

bST

Dextransulfate

1600

100

5

39

5200

6.4

E6

1.6

E6

0.0

2

1/3

$1/3

bST

Heparin

990

176

17

37

3200

3.6

E6

1.2

E6

0.0

2

1/0.7

$1/1

bST

Sucroseoctasulfate

12

0.2

1.5

31

64

2.7

E5

9E4

0.4

NA

NA

bST

Phyticacid

NA

NA

NA

NA

NA

NA

NA

NA

hGH

All

NA

NA

NA

NA

NA

NA

NA

NA

Prot/sacch,proteinsboundpersaccharide.

Figure 2. Representative isothermal titration calori-metry binding isotherm (A) and raw data (B) for pST(3.71 mg/mL) titrated with dextran sulfate (2.2 mg/mL)at 258C using a program of 25 injections of 10 mL.




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number of () charges per mole of ligand. Thisnumber should then represent the heat of bindingper () charge of ligand. The number of proteinsbound per ligand was calculated from the inverseof the stoichiometry. The number of proteinsbound per saccaharide was determined by divid-

ing the number of protein molecules bound perligand by the average number of disaccharides permole of ligand.

Measured enthalpies of interaction for thetitration of polyanions into both pST and bSTwere large and negative. The greatest enthalpiceffect was produced by the interaction of proteinwith the much larger polyanions dextran sulfateand heparin. Dextran sulfate and heparin possessa relatively greater number of negative chargesper molecule than the smaller polyanions phyticacid and SOS. This large number of charges

should result in more electrostatic bindingsites and a greater enthalpic contribution tobinding. Even when normalized on a single chargebasis, the heats of binding of the larger poly-saccharides are greater than the smaller ones(Tab. 1).

Upon interaction, the two components of thebiomolecular complex may change conformationresulting in a more negative or positive DS. Thecomplex itself could become more tightly folded,thereby reducing internal degrees of freedomresulting in a less favorable DS. In contrast,translational and rotational motion may increase,resulting in a more positive entropy contribution.Thermodynamic parameters are state functions,and the measured enthalpy and calculatedentropy are the sum of all processes that occurin the reaction process. Thus, it is inherentlydifficult to assign individual contributions ofbinding phenomenon to the individual thermo-dynamic parameters.

Stoichiometric measurements of the biomole-cular interaction are also possible through the useof ITC. Varying degrees of binding stoichiometrywere measured for complexation of bST and pST

with the polyanions (Tab. 1). The much largerpolyanions dextran sulfate and heparin, bindlarge numbers of protein molecules. A similarresult was observed for aFGF bound to heparin.10

Approximately 1 molecule of pST was bound forevery 3 monosaccharides of dextran sulfate, and1.25 monosaccharides of heparin, 1 bST for every3 monosaccharides of dextran sulfate and 1 ofheparin. Similarly, converting to bound proteinsper ligand results in approximately 40 proteinsper ligand for pSTDS, 25 proteins per ligand for

pSTHeparin, 40 bST molecules per dextransulfate, and 40 bST molecules per heparin chain.

Measurements of the association constants,(Ka), were also compared. As indicated above,an ITC experiment is based on measuring thetotal enthalpy change. Therefore, K values are

only apparent binding constants. Moderate tostrong binding constants were observed for allgrowth hormone polyanion interactions rangingfrom 103106 M1. The strongest affinity wasobserved for the interaction of dextran sulfate andheparin with protein (Tab. 1). Undetectable heatsof binding were observed for hGH as well as phyticacid binding to bST, no doubt reflecting the weakinteraction in these cases.

The major binding event in all of the polyanionprotein interactions of this study is probablyelectrostatic in nature. In some situations, macro-

molecules that bind charged compounds areentropically favored with minor DH values. Thiscan be explained by the release of water moleculessolvating the free-charged compounds. There are,however, examples of electrostatic interactionsbetween macromolecules and ions that areenthalpically driven.1820

Dynamic Light Scattering

DLS measurements were performed to determinethe polyanion/protein complex size and confirmthe stoichiometry estimated by the ITC experi-ments. Light scattering experiments weredesigned to simulate the conditions of the ITCtitrations employing the same concentrations ofmacromolecule and ligand. Binding stoichiometrywas determined based on the saturation point orplateau where no further increase in diameter ofthe polyanion\protein complex was observed. Thestoichiometry based on DLS experiments was ofthe same magnitude as that determined by ITC,$40 proteins per ligand for pSTDS, $25proteins per ligand for pSTHeparin, $40 bST

molecules per dextran sulfate, and 40 bSTmolecules per heparin chain. This value wascalculated by determining the molar ratio ofproteins bound per ligand based on the injectionpoint where saturation is reached (approximatelythe 7th injection for pSTDS) (Fig. 3). Theeffective diameter versus injection number ispresented in Figure 3.

DLS measurements of growth hormones in thepresence of SOS and phytic acid were inconclusiveas to the nature of the stoichiometry. Consistent


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with other methods of analysis, DLS measure-ments of hGH upon titration with polyanionsshowed no significant increase in Stokes radiusupon addition of any ligand over the range ofconcentration employed.

Red Edge Excitation Shifts (REES)

Red edge excitation spectroscopy was employed toprobe the conformational dynamics of the proteinbefore and after binding of polyanions. Fluores-cence emission spectra are usually thought to beindependent of excitation wavelength. Excitationdependent spectra, however, have been observedfor a large number of proteins.2124 Monitoring

the peak position at excitation wavelengths above290 nm will provide information concerning themicroenvironment of these tryptophan residuesas well as their dynamics. Upon excitation, thefluorophores dipole moment changes and solventdipoles can reorient in response. Solvent relaxa-tion may occur faster, slower, or simultaneouslywith emission decay. When relaxation occursslower than or on the same time scale as emission,a complex excitation-dependent emission spec-trum may be observed. When excitation occurs

with low energy quanta (red edge) a smalldistribution of fluorophores will be selectivelyexcited assuming equilibrium is not attained andsolvent relaxation is not complete. The emissionenergies of the fluorophores will necessarily belower than the mean of the distribution and the

emission spectra will be shifted toward longerwavelengths.22

All three growth hormones show a significantred shift upon increasing excitation wavelength(results not shown). Thus, the tryptophan resi-due(s) appear to be positioned in a conformation-ally restricted environment. When polyanions areadded, however, no change in the extent of thepeak position shift is observed, arguing thatligand binding has little or no effect on theproteins conformational dynamics, at least in thevicinity of the single indole side chain.

Aggregation Studies

To further examine the interactions between thethree growth hormones and polyanions, anaggregation-based turbidity assay was developedto produce conditions that could achieve signifi-cant aggregation. This technique monitors thephysical stability of proteins by following thekinetics of aggregation of structurally alteredprotein by measurement of the degree of turbidity(light scattering) of the solution at 360 nm. It was

found that elevated temperatures (378C) and thepresence of DTT induced significant aggregationof two of the proteins. All polyanions producedsome degree of inhibition of bST and pSTaggregation with almost 100% inhibition observedin the presence of dextran sulfate and heparin.

Various ratios of protein to polyanions were tested(Tab. 2) with an example of the aggregationbehavior (pST in the presence of polyanions at a1:1 molar or weight ratios of protein to polyanion)shown in Figure 4. The larger polyanions werepotent inhibitors of aggregation even at very low

molar ratios (Tab. 2). The inhibitory effect ofphytic acid appeared to decrease with an increas-ing weight/molar ratio of phytic acid to protein. Aninhibition assay was not developed for hGH due tothe inherent stability of the protein except underextreme conditions of temperature.

Intrinsic Fluorescence

The effect of polyanions on the thermal stability ofbST, pST, and hGH was monitored using the

Figure 3. Hydrodynamic diameter observed versusinjection number of polyanions for pST employingdynamic light scattering. Each data point represents

the mean () standard error of three samples of proteinligand complex formed by sequential titration of 10 mLof 1.5 mg/mL heparin (~), 2.5 mg/mL dextran sulfate(&), 1 mg/mL phytic acid (), or 1 mg/mL sucroseoctasulfate (^), into 4 mg/mL pST.




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red shift occurs with pST alone and in presence ofall polyanions from $324 nm to 333 nmat approximately 368C when emission peakposition is monitored but no stabilization bypolyanions is seen (Fig. 6C). The relative lightscattering intensity of pST exhibits a markedincrease above 658C when measured alone or inthe presence of phytic acid and SOS (Fig. 6D). Thescattering was dramatically reduced with theaddition of the larger ligands, again indicatingstabilization against aggregation.

hGH was affected the least of the growthhormones by addition of the various polyions.hGH is very stable in solution and shows little tono aggregation upon increasing the temperatureup to 858C. The expected decrease with tempera-ture in the fluorescence intensity of hGH wasobserved both in the presence and absence ofpolyanions (Fig. 7B), although suggestions of asmall transition are evident above 808C. A smallred shift was observed ($3 nm) for hGH in thepresence and absence of polyions (Fig. 7C)

Figure 5. Effect of various polyanions on the thermal unfolding of bST as measured bycircular dichroism and fluorescence spectroscopy. (A) Molar ellipticity at 222 nm of(a) bST, (b) bSTdextran sulfate, and (c) bSTheparin. (B) Fluorescence intensity,(C) wavelength of emission maximum, and (D) light scattering intensity of (^) bST,(&) bSTdextran sulfate, (~) bSTheparin, (o) bSTphytic acid, and () bSTsucrose octasulfate.




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indicative of a structural transition or change inTrp environment due to a direct effect of theligand. The relative light scattering intensity ofhGH in the presence or absence of polyanionsdid not change significantly with temperature(Fig. 7D). Overall, the addition of polyanions doesnot appear to significantly perturb the secondaryor tertiary structure of hGH.

Circular Dichroism

The effect of temperature on the secondarystructure of bST, pST, and hGH in the presenceof various polyanions was analyzed by changes inCD ellipticity at 222 nm. CD measurements showa thermal transition near 608C for bST alone. Inthe presence of the larger polyanions, dextransulfate and heparin, the transition begins at the

lower temperatures of 498C and 568C, respectively(Fig. 5A), suggesting destabilization of bST bythese polyanions. In contrast, the low molecularweight polyanions phytic acid and SOS did notappear to have a significant effect on the Tm valueand hence on the thermal stability of bST(Tab. 3). Dextran sulfate appeared to have amajor destabilizing effect on pST, as demon-strated by a large downward shift in the Tm value(approximately 148C, Fig. 6A). The other poly-anions did not produce a major effect on the

transition temperature upon addition to pST(Tab. 3). As now expected, none of the polyanionsappeared to have a measurable effect on thethermal behavior of hGH (Fig. 7A, Table 3).

DISCUSSIONThe interaction of proteins with polyanions is aphenomenon of increasingly wide interest.9

Despite the importance of the mammalian soma-totropins and their correspondingly extensivebiophysical characterization, with the exceptionof a single report,6 there is little recognition thatthese proteins fall into this category. The biolo-gical significance of such polyanion/protein inter-actions are still unclear although there is growingrecognition that they are important, playing a rolein a number of activities such as storage,

transport, protein folding, and delivery. Unfortu-nately, a direct test of the biological relevance ofthe interactions seen in this article is difficultsince the relatively moderate binding constants ofthe polyanions for the GHs results in rapiddissociation when they are introduced into abiological milieu or cell culture. This would notnecessarily be the case on the surface or insidecells, however.

The potential use of polyanions in therapeuticprotein dosage forms is wide ranging. As previously

Table 3. Transition Midpoint (Tm) of Growth Hormones Alone and in the Presence of Polyanions

Method Polyanion

Transition

Midpoint (8C) bST

Transition

Midpoint (8C) pST

Transition

Midpoint (8C) hGH

Circular dichroism Protein alone 66 74 85Dextran sulfate 53 60 85

Heparin 59 71 85Phytic acid 65 74 85SOS 65 72 85

Fluorescence lmax shift Protein alone 43 35 NDa

Dextran sulfate 45 38 ND

Heparin 42 37 NDPhytic acid 43 36 NDSOS 41 37 ND

ND

Fluorescence intensity Protein alone 63 70 NDDextran sulfate 51 55 NDHeparin 53 53 ND

Phytic acid 58 66 NDSOS 60 65 ND

Errors are approximately 12%.aNot determined.


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mentioned, the thermal stability of many proteinsis increased upon polyanion addition. The additionof polyanions to growth hormones, however,results in decreased Tm values. Decreased transi-tion midpoints do not in themselves necessarilyindicate an inferior formulation. For example, inthis work and others, polyanions have been shownto significantly inhibit protein aggregation. Proteinself-association is one of, if not the major pathwayof physical degradation of protein pharma-

ceuticals. In addition, polyanions may protectmany proteins from mechanical stresses encoun-tered during manufacturing such as shipping,filling, etc. (e.g., agitation shear-stress).

The polyanion/protein interaction seen forbovine and porcine growth hormones results insignificant exothermic (enthalpic) contributions tothe binding equilibrium. The greatest enthalpiceffect was produced by the interaction of proteinwith the larger polyanions dextran sulfate and

Figure 6. Effect of various polyanions on the thermal unfolding of pST as measured by

circular dichroism and fluorescence spectroscopy. (A) Molar ellipticity at 222 nm of(a) pST, (b) pSTdextran sulfate, and (c) pSTheparin. (B) Fluorescence intensity,(C) wavelength of emission maximum, and (D) light scattering intensity of (^) pST,(&) pST dextran sulfate, (~) pSTheparin, (o) pSTphytic acid, and () pSTsucrose octasulfate.




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heparin. Dextran sulfate and heparin possess arelatively greater number of negative chargesthan the smaller polyanions phytic acid and SOS.Therefore, a greater number of charges wouldresult in potentially more electrostatic bindingcontacts and greater enthalpic contribution tobinding despite the well-known entropic contri-bution to electrostatically driven binding inter-

actions. There are examples of electrostaticinteractions between macromolecules and ionsthat are enthalpically driven,1820 includingbinding of a target peptide to the calmodulinbinding site of rabbit smooth muscle myosin lightchain kinase.25 Moreover, the degree of the entr-opy change may be sensitively dependent on thenumber of binding sites on the macromolecule or

Figure 7. Effect of various polyanions on the thermal unfolding of hGH as measuredby circular dichroism and fluorescence spectroscopy. (A) Molar ellipticity at 222 nm of(a) hGH, (b) hGHdextran sulfate, and (c) hGHheparin. (B) Fluorescence intensity,(C) wavelength of emission maximum, and (D) light scattering intensity of ( ) hGH

alone, (&) hGHdextran sulfate (~), hGHheparin, (o) hGHphytic acid, and ()hGH sucrose octasulfate.


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ligand. The enthalpic contribution to the bindingevent increases with increasing number of ()charged protein residues (bSTpolyanion >pSTpolyanion)hGH polyanion). Even on a percharge basis, binding of the larger polyanions isgreater than the smaller one, perhaps suggesting

positively cooperative binding. A large number ofgrowth hormones appear to bind to the largerpolyanions. This may seem surprising at first,however, a number of studies indicate thatmultiple proteins can bind to single polysac-charide chains.10,26,27 Under such conditions,

Table 4. Multiple Sequence Alignments of Human, Bovine, and Porcine Somatotropins Using all DefaultClustal W Parameters

Positively charged residues Histidine, Lysine, and Arginine are highlighted in pink.




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micro-aggregated states are probably formed inwhich multiple proteins bind to extended poly-anion chains which become crosslinked. Dynamiclight scattering experiments demonstrate a het-erogeneous distribution of particle sizes support-ing the existence of such micro-aggregated states

and prohibiting a more rigorous analysis of thethermodynamic data.

In the presence of dextran sulfate and heparin,bST and pST showed a marked reduction in thethermal transition temperature as measured byfluorescence. Interestingly, plots of the fluores-cence intensity (Figs. 5B and 6B) as a function oftemperature indicate different midpoints of ther-mal unfolding as compared to both fluorescencepeak position (Figs. 5C and 6C) and molarellipticity (Figs. 5A and 6A) as a function oftemperature. Fluorescence and CD measure-

ments support the idea that the secondary andtertiary structure of both bST and pST is alteredupon interaction with polyanions. Dextran sulfateand heparin, however, inhibit aggregation of bSTand pST as indicated by a decrease in lightscattering intensity (Figs. 5D and 6D). Further-more, a significant red shift of the emission

maximum is observed with bST and pST indicat-ing increased solvent exposure of the tryptophanresidues. Surprisingly, however, the polyanionsdo not appear to significantly affect the physicalstability or secondary and tertiary structure ofhGH. The fluorescence emission maximum as a

function of temperature for hGH alone and in thepresence of polyanions shifts approximately 3 nmindicating some alteration of the tryptophanenvironments (Fig. 7C), although polyanions donot appear to alter the magnitude of the shift. It isclear, however, that hGH binds heparin, albeitsignificantly more weakly than the other twoproteins.

The mechanism of growth hormone aggregationand inhibition of aggregation by polyanions is notdirectly investigated in this work. It may behypothesized that polyanions bind to a more

native-like conformation of the growth hormonereducing the number and diversity of partiallyunfolded protein states. These partially unfoldedensembles may be prone to self-association.

Another possible explanation for the inhibitionof growth hormone aggregation upon polyanionbinding involves charge repulsion. The high

Figure 8. Calculated electrostatic potential mapsusing the online server protein continuum electro-

statics (http://bioserv.rpbs.jussieu.fr/PCE) of bovinesomatotropin with positive potentials shown in blueand negative potentials shown in red. Calculation para-meters include 4 as the protein internal dielectric, 80 as

the solvent dielectric constant, and 0.1 as the ionicstrength.

Figure 9. Calculated electrostatic potential mapsusing the online server protein continuum electro-statics (http://bioserv.rpbs.jussieu.fr/PCE) of humansomatotropin with positive potentials shown in blue

and negative potentials shown in red. Calculation para-meters include 4 as the protein internal dielectric, 80 asthe solvent dielectric constant, and 0.1 as the ionicstrength.


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negative charge density of polyanions mayprevent aggregation because of unfavorable ener-getics upon self-association. Steric hindrance mayalso prevent protein aggregation. These specificpolyanions are highly flexible and proteinproteininteractions could be blocked by bulky chains.

The temperature dependence of the fluores-cence emission maximum (lmax) and fluorescenceintensity (Fi) are two different physical propertiesthat may be dissimilar depending on the specificchanges in protein structure and fluorophoreenvironment. Therefore, it is not surprisingthat we observe different results from these twomeasurements. The temperature dependence

of lmax and Fi both reflect changes in the Trpenvironment, but they may not necessarilydirectly correlate. For example, Lys residuesmay influence the fluorescence intensity by quen-ching with relatively little effect on the emissionmaximum. Furthermore, changes in the local

dielectric constant and permitivity surroundingindole side chains may influence the lmax to amuch greater extent compared to fluorescenceintensity.

A number of cellular polyanions are thought tomediate protein/receptor interactions. For exam-ple, the interaction of the FGFs with the FGFRsrequires heparan sulfate glycosaminoglycans.

Figure 10. Molecular surface representations of (A, B) bovine somatotropin,(C) human somatotropin bound to two receptors, and (D) human somatotropin alone

with the residues histidine, lysine, and arginine shown in blue and labeled. Visualmolecular dynamics (VMD) was used to produce the surface representations. ProteinData Bank structures 1bst, 1hgu, and 3hhr were used for bovine somatotropin, humansomatotropin, and the human growth hormone receptor complex, respectively.




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Heparan sulfate binds both the receptor and FGFfacilitating the formation of a large FGF/FGFR/HSPG complex essential for signaling.28,29 Manyexamples exist of this phenomenon.8,30,31 Thesites of polyanion binding have in many casesbeen determined from X-ray crystallography and

NMR studies and the general conclusion is thatpolyanions bind to clusters of basic amino acids onthe surfaces of such proteins. In some instances,receptor interaction with the target proteinresults in the alignment of a contiguous patchof basic amino acid residues facilitating thedocking of polyanionic proteoglycans. In additionto mediating cell signaling, cellular polyanionscan interact with proteins in a wide range ofbiologically functional situations. Heparan sulfateproteoglycans regulate the formation of concen-tration gradients, and localize the response and

strength of signaling of many proteins includinggrowth factors and chemokines.8,30,32 It is possiblethat growth hormones interact with cellularpolyanions in a functionally similar context tothat of the fibroblast growth factors, manychemokines and other proteins, but this remainsto be directly demonstrated.

A multiple sequence alignment of bovine,porcine, and human somatotropin is shown inTable 4 with the basic amino acid residueshighlighted. The number of lysine, arginine,and histidine residues is greatest for bST,28

followed by pST27 and hGH.22 This decrease inthe polycationic nature of hGH compared to theother two proteins can also be seen on the surfaceof hGH as observed by electrostatic surfacerepresentations (Figs. 8 and 9). Two patches ofbasic residues in the surface of pST and bSTinvolving Lys30 and Arg34, as well as Arg108 andLys112 are absent in hGH. These observationsmay help explain the reduced interaction of hGHwith the polyanions studied. An integratedbioinformatics software program, the Sting Mil-lenium Suite,3335 was used to further probe thenature of GH basic residues by determining

accessible surface areas and internal residuecontacts. The solvent accessible surface area isquite high for the basic residues missing in hGHwith values of 124.5, 157.2, 160.5, and 174.7 forK30, R108, R34, and K112, respectively. Potentialpolyanion binding hot spots are shown inFigures 810 and it appears that bST and hGHmay share a common potential polyanion bindingsurface. Common clusters of basic residuesinclude R17, 166, 177,183 and K167, 171 of bSTand R16, 167,178,183 and K168, 172 of hGH. The

hGH receptor complex, as revealed by crystal-lographic studies,36 suggests a potential poly-anion binding site across the face of the complexinvolving a patch of basic amino acid residues(Lys121,167 and Arg 71,126,183,213) (Fig. 10CD).

In vivo, one growth hormone molecule binds and

dimerizes two receptor molecules in a sequentialfashion,3742 initially via a high affinity event thenthrough a relatively low affinity allostericallycoupled interaction.43Although the mechanism ofGHR activation is still debated, it has been shownthat alignment of the receptor molecules may beimportant in signaling and that dimerizationalone is insufficient to activate GHR.44,45 Thus,polyanions might facilitate the formation andalignment of the hGH receptor complex in asimilar fashion to the FGF/FGFR/HSPG complexby binding simultaneously to both proteins.

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growth hormone stability paper

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