influence of cyclodextrin ring substituents on folding-related aggregation of bovine carbonic...

Influence of cyclodextrin ring substituents on folding-relatedaggregation of bovine carbonic anhydrase

Loretta Sharma1 and Ajit Sharma2

1Science Department, Davenport University, Midland, MI, USA; 2Department of Chemistry, Central Michigan University, Mt Pleasant,

MI, USA

A common obstacle to proper renaturation of an unfolded

protein is aggregation, an intermolecular side reaction of

immense importance in biotechnology and in the patho-

genesis of several neurodegenerative diseases. Cyclic

sugars known as cyclodextrins have been used as protein-

folding aids. The effect of cyclodextrin chemistry on

aggregation and refolding of carbonic anhydrase was

evaluated in this study. Size-exclusion HPLC showed that

cyclodextrins inhibit aggregate formation without interfer-

ing with the correct renaturation of carbonic anhydrase.

PAGE of refolded enzyme provides further evidence of

inhibition of folding-related aggregation by natural and

chemically modified cyclodextrins. Although the amount of

aggregate formed and recovery of active enzyme was

dependent on cavity size, the nature of the chemical

substituents found on the rims of the sugar molecule seems

to play a more important role in cyclodextrin-assisted

refolding of carbonic anhydrase. In general, neutral or

cationic cyclodextrins with small cavities were found to be

better folding aids than anionic cyclodextrins with larger

holes. Although the exact prediction of the effect of a

cyclodextrin substitution on protein refolding is not

possible at present, these results clearly show that modified

cyclodextrins can be designed that effectively inhibit

protein aggregation.

Keywords: aggregation; carbonic anhydrase; cyclodextrin;

refolding; renaturation.

Natural cyclodextrins are water-soluble, conical, cylinder-shaped, cyclic glucose oligosaccharides formed by theaction of bacterial enzymes on starch (Fig. 1). There arethree common types of natural cyclodextrin depending onhow many sugar units are present: a-cyclodextrin has sixglucose units, b-cyclodextrin has seven, and g-cyclodextrinhas eight. The salient characteristic of a cyclodextrinmolecule is the presence of a central `cavity' or `hole'which provides an excellent resting site for hydrophobicmolecules of appropriate dimensions. On one edge of thiscavity are the secondary hydroxy groups, while all theprimary hydroxy groups are located on the other rim. Themore glucose units in the cyclodextrin circle, the largerthe cavity. Cavity depth for a-cyclodextrin, b-cyclodextrinand g-cyclodextrin is < 7.9 AÊ while their diameters areapproximately 5, 6 and 8 AÊ , respectively [1]. Thus cyclo-dextrins act as molecular carrier hosts for nonpolar guestsin an aqueous environment. This property of cyclodextrinshas extensive applications whenever hydrophobic mol-ecules are used in polar environments such as water.Cyclodextrins have been used successfully in pharma-ceutical, food and flavour industries, biotechnology,cosmetics, diagnostics and waste treatment [1].

Protein aggregation is an important phenomenon inbiotechnology and human disease [2]. In biotechnology, a

critical step in the recovery of active recombinant proteinfrom solubilized inclusion bodies is protein refolding [3,4].Because of formation of intermediate(s) with exposedhydrophobic residues, competing reactions leading toaggregation result in low recovery of functionally activeprotein [3±5]. In medicine, the list of diseases associatedwith protein misfolding and aggregation keeps increasing.Hungtington's, Alzheimer's and Parkinson's diseases areexamples of human diseases in which the deposition ofprotein aggregates is responsible for their pathology [2].

As aggregation is an intermolecular reaction, one prac-tical way of overcoming this obstacle in biotechnology is toperform protein refolding under low protein concentrations.Although this can lead to substantial improvements in yield,the resulting large volumes make it difficult and expensiveto purify the active protein. Improved protein-folding yieldshave been obtained by using various dilution protocols andthe addition of buffer additives or folding aids [3±5]. Theability of cyclodextrins to rapidly form reversible, non-covalent inclusion complexes with hydrophobic moleculesor parts of molecules has been used in protein-foldingtechnology [6,7]. Cyclodextrins have many advantages asbuffer additives for protein refolding. They are biocom-patible, nontoxic, stable and relatively inexpensive. Theynot only enhance protein folding, but also act as anti-aggregating agents for native proteins that have a tendencyto aggregate in solution [8,9]. In addition, cyclodextrinscan stabilize proteins prone to degradation during storage.Improved yields of active carbonic anhydrase were obtainedwhen guanidine hydrochloride (GdnHCl)-denaturedenzyme was refolded in the presence of cyclodextrins [6].Recovery of active protein was dependent on the concen-tration of cyclodextrin as well as its ring size. Turbidimetricstudies suggest that the cyclodextrin-enhanced protein

Eur. J. Biochem. 268, 2456±2463 (2001) q FEBS 2001

Correspondence to A. Sharma, Department of Chemistry, Dow 346,

Mt Pleasant, MI 48859, USA. Fax: 1 517 774 3883,

E-mail: [email protected]

Abbreviations: CMCD, chemically modified cyclodextrin; GdnHCl,

guanidine hydrochloride.

Enzyme: carbonic anhydrase (EC 4.2.1.1).

(Received 31 January 2001, accepted 26 February 2001)

folding may be partly due to the ability of cyclodextrins toprevent aggregate formation during protein refolding [6].These studies on carbonic anhydrase demonstrated theability of cyclodextrins to enhance protein-refolding yieldsin vitro.

The main objective of this investigation is to shed furtherlight on the antiprotein aggregation behavior of these cyclicoligosaccharides. Inhibition of folding-related aggregateformation was followed by PAGE and size-exclusionchromatography with light-scattering detection. Cyclo-dextrins with various ring substituents [referred to aschemically modified cyclodextrins (CMCDs)] were evalu-ated to determine which part of the cyclodextrin molecule isessential for its antiaggregation property: the central cavityor the rims of hydroxy groups or both. This structure±activity relationship should help us to understand theinteractions between the rapidly folding polypeptide chainsof carbonic anhydrase with the 20±30-fold smaller oligo-saccharide hosts. Such information will be useful in design-ing improved cyclodextrin-based anti-(protein-aggregation)agents.

M A T E R I A L S A N D M E T H O D S

Carbonic anhydrase II (electrophoretically purified frombovine erythrocytes, formerly designated carbonic anhy-drase B) was purchased from Sigma Chemical Co. (StLouis, MO, USA). PAGE of the native protein showed amajor band and a faster migrating minor band when the gelwas stained with either Coomassie blue or silver. Trehalose,dextrose and maltohexaose were also obtained from Sigma.a-cyclodextrin, hydroxypropyl b-cyclodextrin, g-cyclodex-trin, quaternary ammonium b-cyclodextrin, hydroxyethylb-cyclodextrin, tertiary amine carboxymethyl b-cyclodex-trin, tertiary amine b-cyclodextrin, methyl b-cyclodextrinand quaternary ammonium a-cyclodextrin were obtainedfrom Cerestar USA, Inc (Hammond, IN, USA).a-Cyclodextrin phosphate, 2-hydroxypropyl a-cyclodextrin,

b-cyclodextrin phosphate, carboxymethyl a-cyclodextrinand carboxymethyl b-cyclodextrin were obtained fromPharmatec, Inc (Alachua, FL, USA). Acetyl b-cyclodextrinand acetyl g-cyclodextrin were from Wacker BiochemicalCorporation (Adrian, MI, USA). GdnHCl was obtainedfrom Life Technologies, Inc. (Gaithersburg, MD, USA).

Protein concentration

Protein concentration of native carbonic anhydrase in50 mm Tris/sulfate at pH 8.5 was determined by measuringits A280 using an absorption coefficient of 1.83 mg proteinper mL per cm and a molecular mass of 30 kDa [6].

PAGE

Nondenaturing discontinuous PAGE was carried out usinga 10% separating gel (pH 8.8) without a stacking gel.Preparation of the slab gel and separation were performedwith the Dual Mini-Vertical Slab Gel Electrophoresis Unitfrom Sigma. Gels were stained with Coomassie blue.Carbonic anhydrase was denatured at room temperature for3 h in a fresh solution of 5 m urea made in 50 mm glycinephosphate buffer, pH 2.3. Refolding was performed byrapid mixing of 1 vol. denatured carbonic anhydrase with14 vol. cold (4 8C) 50 mm sodium phosphate buffer, pH 8.5(containing the cyclodextrin additive). Then 3 vol. of therefolded protein was mixed with 1 vol. cold 4 � sampleloading buffer [100 mm Tris/HCl, pH 8.0, 40% glyceroland 0.01% (w/v) bromophenol blue], and the mixture wasimmediately applied to the gel. Electrophoresis wasperformed at 240 V at 4 8C.

Size-exclusion chromatography/laser light scattering

Size-exclusion chromatography was carried out at roomtemperature on a Superdex 200 HR 10/30 column(Pharmacia), which separates proteins with molecularmasses in the 10- to 600-kDa range. The buffer used was20 mm Hepes/100 mm KCl/1 mm EDTA, pH 8.0. Thecolumn was connected on-line to three detectors: UVabsorbance (model 773 variable wavelength; KRATOS),light scattering (DAWN±DSP; Wyatt Technology, SantaBarbara, CA, USA), and refractive index (OPTILAB DSP:Wyatt Technology) detectors. Flow rate was 0.4 mL´min21.All samples were filtered through a 0.22-mm filter (low-binding Duraporew membrane; Millipore). Molecular masswas determined by (a) a `two-detector method' from acalibration curve obtained for a series of protein standardsin which the relationship between molecular mass and theratio of the light scattering and refractive index signals wereplotted, or (b) solving the equation that relates the excessscattered light, measured at several angles, to the concen-tration of solute and the weight-average molar mass(ASTRA software).

Esterase activity of carbonic anhydrase

The catalytic activity of carbonic anhydrase was determinedby measuring its hydrolysis of the substrate, p-nitrophenolacetate (50 mm Tris/sulfate, pH 7.5; concentration of sub-strate in assay mixture was 1 mm), forming p-nitrophenol,which was monitored at 400 nm and 25 8C in a double-beam spectrophotometer (PerkinElmer Lambda 20). Assays

Fig. 1. Structure of a-cyclodextrin. In chemically modified cyclo-

dextrins some of the hydroxy groups are converted into other

functional groups.

q FEBS 2001 Effect of cyclodextrin substituents on protein aggregation (Eur. J. Biochem. 268) 2457

were performed at pH 7.5 because blank rates increaseappreciably at higher pH. At the concentrations used in thisinvestigation, there was no significant hydrolysis of thesubstrate by any of the cyclodextrins used in this study.

Carbonic anhydrase denaturation/renaturation

Except for the electrophoresis experiment, carbonic anhy-drase was denatured by overnight incubation in 6 mGdnHCl in 20 mm Tris/sulfate, pH 8.5, at 25 8C. Refoldingof the enzyme was achieved by rapid dilution in renatura-tion buffer consisting of 50 mm Tris/sulfate, pH 8.5, with orwithout additive.

Turbidimetric measurements

Turbidimetric analysis of protein aggregation was per-formed at 25 8C on a PerkinElmer Lambda 20 spectro-photometer. The apparent absorbance obtained on additionof denatured carbonic anhydrase to renaturation bufferfollowed by rapid mixing was measured at 350 nm in aquartz cell of 1-cm path length.

R E S U L T S

A large number of cyclodextrin derivatives have beenreported [10]. In this study, we selected neutral, cationic,anionic and amphoteric cyclodextrins, which are commer-cially available in purified forms and which possess variousring substituents (Fig. 1). Their effect on carbonic anhy-drase refolding was compared with the natural cyclo-dextrins, a-cyclodextrin and g-cyclodextrin (b-cyclodextrinwas not used because of its poor aqueous solubility) aswell as with noncyclic sugars (glucose, trehalose andmaltohexaose).

The aggregation kinetics of carbonic anhydrase in thepresence of various CMCDs was monitored by turbidi-metry. CMCD concentration was 50 mm (about 6%, w/v)for cyclodextrins of known molecular mass and 6% (w/v)for those of unknown molecular mass. Denatured carbonicanhydrase was added to renaturation buffer (50 mm Tris/sulfate, pH 8.5, with or without cyclodextrin) in a quartzcuvette. The final protein concentration in the cuvette was30 mg´mL21. Light scattering due to formation of proteinaggregates caused an apparent increase in absorbance,which was monitored at 350 nm. At this wavelength, thereis no absorption by the protein, cyclodextrin or any otheringredients in the cuvettete. The basic curve obtained formost CMCDs was similar to those of controls (without anycyclodextrin present) and parent cyclodextrin (e.g. a or g).Maximum apparent turbidity reaches a plateau within1±2 min (Fig. 2). The only exceptions were the carboxy-methylated cyclodextrin derivatives (carboxymethyla-cyclodextrin and carboxymethyl b-cyclodextrin), whereaggregation increased gradually with time (Fig. 2).

The antiaggregation properties of various cyclodextrinswere compared by estimating the amount of proteinaggregate formed on renaturation of denatured carbonicanhydrase. A semiquantitative measurement of aggregateamount was obtained by measuring the plateau absorbancesobtained (Fig. 3). In general, the larger the apparentabsorbance, the greater the amount or size of aggregateformed. As above, denatured carbonic anhydrase was added

to refolding buffers (final protein concentration in thecuvette was 30 mg´mL21) containing cyclodextrins or othersugar additives (additive concentration was 50 mm or 6.5%in all cases). After 1 h, absorbance of duplicate tubes wasmeasured. It is clear from Fig. 2 that the cavity dimensionsas well as the presence of chemical substituents on thecyclodextrin ring have a profound effect on its interactionwith the folding polypeptide chains of carbonic anhydrase.A number of cyclodextrins were effective antiaggregationagents with average absorbances less than 70% that ofcontrol. These include the parent cyclodextrin a-cyclo-dextrin as well as a number of CMCDs includingquaternary ammonium a-cyclodextrin, hydroxypropyla-cyclodextrin, acetyl b-cyclodextrin, acetyl g-cyclo-dextrin, b-cyclodextrin phosphate and tertiary aminecarboxymethyl b-cyclodextrin. On the other hand, twocyclodextrins actually promoted aggregation during car-bonic anhydrase refolding. Both were carboxymethylatedderivatives (carboxymethyl a-cyclodextrin and carboxy-methyl b-cyclodextrin, which gave absorbances < 1.6-foldand sixfold, respectively, over control). For comparison, thenoncyclic six-membered glucose oligosaccharide, malto-hexaose, and other sugars such as trehalose and glucose,showed turbidities similar to or higher than controls. It isevident from Fig. 3 that for a given ring size such asa-cyclodextrin, the presence of negatively chargedgroups such as phosphate or carboxymethyl decreased the

Fig. 2. Aggregation kinetics of carbonic anhydrase. Denatured

carbonic anhydrase was added to renaturation buffers [with 50 mm

or 6.5% (w/v) cyclodextrin or without additive] in a quartz cuvette. The

solution was rapidly mixed and placed in a spectrophotometer.

Absorbance was monitored at 350 nm. Final protein concentration in

the cuvette was 30 mg´mL21. In the absence or presence of most

cyclodextrins, the shape of the curve obtained is shown as `A'. In the

presence of carboxymethyl cyclodextrin derivatives, the curve obtained

was `B'. Values are means of duplicate determinations. The coefficient

of variation in all cases was less than 20%.

2458 L. Sharma and A. Sharma (Eur. J. Biochem. 268) q FEBS 2001

effectiveness of a-cyclodextrin as an antiaggregation agent.Within experimental error, the presence of neutral orpositively charged substituents on the cyclodextrin ring,such as hydroxypropyl or quaternary ammonium groups,did not have any deleterious effect on the antiaggregationability of the parent a-cyclodextrin.

To assess the effect of cyclodextrins on the recovery ofcorrectly folded protein, it was assumed that misfoldedprotein was functionally inactive [3]. The catalytic activityof refolded protein was determined after renaturation ofdenatured protein in buffer (Tris/sulfate, pH 8.5) containing50 mm a-cyclodextrin or g-cyclodextrin or 6% (w/v)CMCD. Final protein and GdnHCl concentrations were1 mg´mL21 and 0.24 m, respectively. Renaturation wasallowed to occur for 24 h for maximum effect [6]. Theamount of catalytically functional carbonic anhydrase wasdetermined by its esterase activity with p-nitrophenylacetate. The results of this experiment are summarized inFig. 4. When refolding was performed in the absence ofcyclodextrin (control), about 25% of enzymatic activity wasrecovered. Increased yields were obtained with the parentcyclodextrins, a-cyclodextrin and g-cyclodextrin, as well asa number of CMCDs including hydroxypropyl a-cyclo-dextrin, acetyl b-cyclodextrin, acetyl g-cyclodextrin,b-cyclodextrin phosphate, tertiary amine carboxymethylb-cyclodextrin and quaternary ammonium a-cyclodextrin.Of these, the highest refolding yields were obtained with

the acetylated cyclodextrins, acetyl b-cyclodextrin andacetyl g-cyclodextrin. Refolding carbonic anhydrase in thepresence of carboxymethylated cyclodextrin derivatives(carboxymethyl a-cyclodextrin or carboxymethyl b-cyclo-dextrin) resulted in complete loss of catalytic activity.However, the amphoteric carboxymethyl b-cyclodextrin(tertiary amine carboxymethyl b-cyclodextrin) showed theopposite effect. When refolding was performed in thepresence of buffer containing the noncyclic glucose oligo-saccharide, maltohexaose, the recovery of activity wassimilar to that of the control. Other sugars such as trehaloseand glucose also gave yields similar to that of the control. Acomparison of the amount of aggregate formed with thepercentage recovery of active protein (Figs 3 and 4) showsan inverse relationship. Increased aggregate formation isassociated with low refolding yields.

To confirm aggregate formation and recovery of nativeenzyme structure with cyclodextrin-assisted carbonic anhy-drase refolding, size-exclusion chromatography and PAGEexperiments were carried out. Carbonic anhydrase, dena-tured overnight in 6 m GdnHCl was refolded at final proteinand GdnHCl concentrations of 1 mg´mL21 and 0.6 m,respectively. The samples were refolded for 24 h andthen analyzed by HPLC size-exclusion chromatography(Fig. 5). The trace shown (Fig. 5A) is from the light-scattering detector at 90 8 (the highest scattering signal). Inthe tracing, a peak at < 9 mL, which was due to dust

Fig. 4. Effect of CMCDs on enzyme recovery. The catalytic activity

of refolded carbonic anhydrase was determined by refolding denatured

protein in buffers containing 50 mm or 6.5% (w/v) cyclodextrin. Final

protein and guanidinium concentrations were 1 mg´mL21 and 0.24 m,

respectively. Renaturation was allowed to occur for 24 h for maximum

activity. The percentage recoveries shown are means of duplicate

determinations. The coefficient of variation in all cases was less than

15%. CD, Cyclodextrin.

Fig. 3. Effect of CMCDs on carbonic anhydrase aggregation.

Denatured carbonic anhydrase was refolded as in Fig. 2. Duplicate

determinations of A350 were made after 1 h incubation and averaged.

Note that the mean absorbance obtained with carboxymethyl b-

cyclodextrin (i.e. 0.4) exceeded the limits of the y-axis. The coefficient

of variation in all cases was less than 20%. CD, Cyclodextrin.


impurities in the sample/injector (this was not a proteinpeak because the refractive index signal indicated that thislight scattering peak did not have any significant massassociated with it), was corrected for in all samples. Nativeenzyme (N, Fig. 5A) showed a major peak eluted at< 17 mL under these conditions. The molecular mass ofthe native protein was estimated to be 28.8 and 28.7 kDa bythe `two-detector' and ASTRA methods, respectively. Theweight-average molar mass did not depend on proteinconcentration, and this peak was monodisperse and con-tained a monomer of the protein. Denatured carbonicanhydrase that refolded without any additive (C, Fig. 5A)also showed a major component (the correctly foldedprotein) which was eluted at < 17 mL (Fig. 5A). This peakwas monodisperse and its molecular mass was estimated tobe 29.3 and 28.4 kDa by the `two-detector' and ASTRAmethods, respectively. In addition, a very broad peak waseluted at < 11 mL. The molecular mass of this peak wasdifficult to evaluate because the refractive index signalproduced by the component was very weak. Based onelution of BSA monomer (molecular mass 67 kDa) at< 15.5 mL and dimer at < 13.8 mL under similar condi-tions, the carbonic anhydrase peak at 11 mL probablyrepresents a mixture of oligomeric species larger than130 kDa. When carbonic anhydrase was refolded in thepresence of a-cyclodextrin (T, Fig. 5A), the size distri-bution of aggregates became narrower, with a shift of theoligomeric protein peak from < 11 mL to < 13 mL. Once

again, the major component representing the correctlyfolded protein was eluted at < 17 mL and its molecularmass was estimated to be 28.4 and 28.0 kDa by the `two-detector' and ASTRA methods, respectively. The A280

tracings for these samples, shown in Fig. 5B, show similarobservations.

The effect of cyclodextrins on folding-related aggregateformation was also evaluated by PAGE (Fig. 6). Carbonicanhydrase samples renatured in various cyclodextrin bufferswas subjected to nondenaturing PAGE. In order to `trap'aggregates, denaturation was carried out with urea, andrefolding and electrophoresis were performed in the cold[11]. Urea-denatured enzyme was refolded to 1 mg´mL21

protein and 0.3 m urea in buffers containing variouscyclodextrin additives (Fig. 6). The native protein (lane 1,Fig. 6) showed a major band as well as a faster movingminor band. The occurrence of carbonic anhydrase II astwo bands on electrophoresis gels has previously beenreported [12,13]. Both fractions are enzymatically active[12]. Enzyme refolded without any cyclodextrin additiveshowed very little native protein (controls, lanes 2 and 7).Best recoveries of native protein were obtained witha-cyclodextrin (lane 6) and g-cyclodextrin (lane 3). Thepositively charged a-cyclodextrin derivative producedsome recovery (lane 5) while the negatively chargedcarboxymethyl a-cyclodextrin showed the lowest amount

Fig. 5. HPLC/light scattering detector tracing (A) and UV 280 nm

tracing (B) of refolded carbonic anhydrase. Unfolded carbonic

anhydrase was renatured in buffer (50 mm Tris/sulfate, pH 8.5) without

any additive (C) or with 100 mm a-cyclodextrin (T), at final

[GdmHCl] and [protein] of 0.6 m and 1 mg´mL21, respectively.

Chromatography was performed after 24 h of renaturation at room

temperature. Native enzyme was also run at a [protein] of 1 mg´mL21

(N).

Fig. 6. Nondenaturing PAGE of refolded carbonic anhydrase.

Urea-denatured carbonic anhydrase was refolded in the presence of

various cyclodextrins (cyclodextrin concentration in each case was

50 mm or 6.5%, w/v). Each lane contained 20 mg protein. Lane

assignments were g-cyclodextrin (3), carboxymethyl a-cyclodextrin

(4), quaternary ammonium a-cyclodextrin (5), a-cyclodextrin (6) and

control, without any additive (2 and 7). Native enzyme is shown in lane

1. Details of the experiment are described in Materials and methods.


of correctly refolded protein (lane 4). Large aggregates thatbarely enter the gel are also clearly visible, especially withenzyme refolded with g-cyclodextrin and a-cyclodextrin(lanes 3 and 6, respectively). Aggregates formed in controlsamples as well as the other cyclodextrin-refolded carbonicanhydrase were probably so large that they could not enterthe gels and were washed away during gel processing andstaining.

D I S C U S S I O N

Previous reports on refolding of carbonic anhydrase [14,15]have shown that, like many other proteins, it tends toaggregate with increasing protein concentration and at lowdenaturant concentrations (e.g. less than 1 m GdnHCl).Refolding of carbonic anhydrase has been reported toproceed through an intermediate species, the first observedintermediate that contains exposed hydrophobic clusters.Under appropriate conditions, this intermediate may formdimers and micron sized aggregates, leading to diminishedrecovery of active protein [15].

A number of folding enhancers have been described [16].These include nondenaturing concentrations of chaotropicagents (urea and GdnHCl), polymers [poly(ethylene glycol),poly(amino acids)], detergents (Triton X-100, lauryl mal-toside, Chaps), arginine and small organic molecules(formamide, acetamide, alcohols). Most of these additiveshave been shown to suppress aggregation during proteinrefolding. The mechanism of action of additives on proteinfolding is unclear, and therefore predicting whether acertain additive will work for a given protein is currentlynot possible.

We have previously demonstrated the use of cyclicsugars known as cyclodextrins as protein-folding aids [6].Recently, a-cyclodextrin has been successfully utilized asan artificial chaperone for refolding the oligomeric protein,phosphofructokinase-1 [7]. Very little is known about theinteraction of cyclodextrins with biopolymers such asproteins. Several factors affect the complexation of guests(in this case, the amino-acid residues of a polypeptide) withcyclodextrin, such as the size of the cyclodextrin cavity andvarious chemical forces. A phenyl ring, for example, fitstightly into the cavity of a-cyclodextrin, resulting instronger binding force. On the other hand, in the case ofb-cyclodextrin or g-cyclodextrin, the cavity size is too largefor a snug fit, leading to weaker guest±cyclodextrininteraction [17]. Substitutions on the guest can also havea significant effect on the binding [17]. However, for theeffective use of cyclodextrins as folding aids and generalanti-(protein-aggregating) agents, a practical way to altercyclodextrin±protein binding is to chemically modify theoligosaccharide host. Substitutions of the hydroxy groups incyclodextrins can lead to enhanced or decreased binding ofguest molecules [18] and this therefore seems an attractiveapproach to designing cyclodextrin derivatives that couldalter the physicochemical characteristics of proteins.

Electrophoresis and chromatography results (Figs 5 and6) confirm inhibition of aggregate formation by cyclo-dextrins shown by turbidimetry. Size-exclusion HPLC clearlyshows that addition of a-cyclodextrin to the refolding bufferinhibits the formation of higher-order aggregates withoutinterfering with the correct regeneration of the native protein(Fig. 5). Cyclodextrins effectively suppressed aggregation.

This effect was more pronounced, especially at lowerGdnHCl concentrations in the refolding buffer (Fig. 5A).However, this phenomenon is dependent on the nature ofsubstituents on the cyclodextrin molecule. The presence ofnegatively charged carboxymethyl groups on the cyclo-dextrin ring actually enhanced aggregation, resulting incomplete loss of functional protein (Fig. 4). This carboxy-methyl-cyclodextrin-induced aggregation was also observedwith native carbonic anhydrase (results not shown), althoughto a much lesser extent. It is possible that the negativelycharged carboxymethyl groups on the oligosaccharide hostmay form strong electrostatic linkages with positivelycharged sites on carbonic anhydrase (or its zinc cofactor). Itis interesting to note that the effect of the negativelycharged carboxymethyl group was effectively neutralizedby the presence of the positively charged tertiary aminegroup on the cyclodextrin ring (Figs 3 and 4). The effect ofcharged derivatives of b-cyclodextrin with neutral andcharged guests was studied by Matsui and coworkers, whoshowed that electrostatic attractive or repulsive forces couldsignificantly alter cyclodextrin±guest binding [18]. Themajor forces involved in cyclodextrin±guest complexationinclude hydrophobic interaction, dipole/dipole interactions,London dispersion forces and van der Waals forces. If thebinding force between the additive and the folding poly-peptide chain is too strong, resulting in failure to release theguest when desired, then part of or the entire additive maybe trapped. This was observed with detergent-assisted pro-tein refolding [19] and may be the case with the carboxy-methylated cyclodextrins. It is important to note that not allnegatively charged cyclodextrins behaved in the samefashion. Aggregate formation and refolding yields obtainedwith a-cyclodextrin phosphate were significantly differentfrom those obtained with carboxymethyl a-cyclodextrin.There was even a more dramatic difference betweenb-cyclodextrin phosphate and carboxymethyl b-cyclo-dextrin. The former was an effective folding aid whereasthe latter showed exactly the opposite effect. Neutral orcationic cyclodextrins always gave lower aggregation andyields higher than the control without any additive (Figs 3and 4). For a fixed cavity dimension, recovery of cata-lytically active enzyme was dependent on the substituentson the cyclodextrin ring. For example, in the case ofa-cyclodextrin (Fig. 4), yields ranged from 0 to 10% for thenegatively charged derivatives to over 40% for the neutralor positively charged derivatives. Whether this trend isunique to this enzyme remains to be seen. These obser-vations clearly demonstrate the impact of the substituentson the cyclodextrin ring on its aggregation-inhibition property.

Interactions between cyclodextrins and the foldingpolypeptide chains of carbonic anhydrase therefore seemto be quite specific. When the guest is a macromoleculewith several potential binding sites, such as a polypeptidechain, a special type of cyclodextrin complex is formed.Complexation of the side groups gives a comb-like struc-ture while complexation of the polymer chain results in anecklace-type structure. With biopolymers such as proteins,a comb-like structure is more conceivable. Likely bindingsites for complex formation in proteins are aromatic aminoacids, which are known to form weak complexes (bindingconstants , 10 m21 with a-cyclodextrin and b-cyclodextrin[20]. Formation of inclusion complexes between b-cyclo-dextrin and peptides containing aromatic amino-acid residues


has been confirmed by electrospray ionization MS [21].NMR and fluorescence techniques have also been used todemonstrate the weak interaction of cyclodextrins withhydrophobic amino-acid residues in the proteins, buserelinacetate and insulin [22]. Cyclodextrins are also known toinhibit a variety of degradation processes of peptides inaqueous solutions [22]. All these studies are consistent withweak complex-formation between cyclodextrin and exposedhydrophobic residues on the polypeptide chain. It istherefore reasonable to assume that such interactions arelikely to occur in protein refolding, because most of theburied hydrophobic residues of the polypeptide becomeexposed in the unfolded state. The importance of thecyclodextrin cavity in cyclodextrin-assisted refolding ofcarbonic anhydrase was previously demonstrated [6] and isalso evident from the results shown in Figs 3 and 4.Refolding of the enzyme in the presence of the cyclic formof the hexose (i.e. a-cyclodextrin) gave over 50% yieldof active protein while the linear form of the hexose(maltohexaose) at a similar concentration (50 mm) resultedin less than half the yield. Competitive studies withphenylalanine also indirectly showed the importance ofthe cyclodextrin cavity in cyclodextrin-assisted carbonicanhydrase [6]. In addition, under identical experimentalconditions, a-cyclodextrin, with a smaller cavity, consis-tently gave lower aggregation and higher refolding yieldsthan g-cyclodextrin which has a larger cavity. However, thepresence of appropriate substituents on the cyclodextrinring seem to be more important than cavity size in deter-mining its protein-refolding ability. For example, acetylg-cyclodextrin gave lower aggregation and better yieldsthan a-cyclodextrin (Figs 3 and 4). One reason for this maybe the influence of ring substituents on accessibility of theguest to the cyclodextrin cavity [17].

The results presented indicate that both the cavity and theenvironment around the two rims have important roles incyclodextrin-enhanced refolding of carbonic anhydrase. Atpresent, insufficient knowledge prevents the exact pre-diction of the effect of a cyclodextrin substitution onprotein refolding. It is interesting to note that the acetyl-cyclodextrins (acetyl-b-cyclodextrin and acetyl-g-cyclo-dextrin) gave the highest refolding yields with carbonicanhydrase (Fig. 4). The presence of groups in which acarbon is connected to an electronegative atom (such as O,N or S) by a double bond in an additive seems to make it agood anti-(protein-aggregating) agent. Examples of thesegroups include C�N found in the guanidino group ofGdnHCl and arginine, C�O found in urea, acetylcyclodex-trins, and acetoamide, and S�O found in dimethylsulfoxide [23]. The large variations obtained in aggregateformation with various cyclodextrins suggest that appro-priate chemical modifications of the substituents on thecyclodextrin ring should allow the design of new cyclo-dextrin-based anti-(protein-aggregating) agents. Furtherwork with other CMCDs and proteins should shed morelight on this complex interaction between cyclodextrins andpolypeptide chains.

A C K N O W L E D G E M E N T S

This work was supported in part by REF no. 62732 and 62733 from the

State of Michigan and FRCE Grants from CMU. The size-exclusion

chromatography/light scattering work was carried out by Dr E.

Folta-Stogniew (Keck Biotechnology Laboratories) for which we are

most grateful.

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