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Isoelectric Immobiline membranes

5 References [l] Wada, A,, Nature 1987, 325, 771-772. [2] Martin, W. J. and Davies, R. W., BiolTechnology 1986, 4, 890-895. [3] Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connel, C. R.,Heiner, C.,Kent, S. B.H. and Hood,L.E., Nature 1986,

321,674-679. [4] Ansorge, W., Sproat, B. S., Stegemann, J. and Schwager, C., J. Biochem. Biophys. Methods 1986, 13, 315-323. [5] Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J.,Cocuzza,A. J., Jensen, M. A. and Baumeister, K., Science 1987,238, 336-341. [6] Kambara, H., Nishikawa, T., Katayama, Y. and Yamaguchi, T., Bio/ Technology 1988, 6, 816-821.

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[7] Britten, R. J., Baron, W. F., Stout, D. B. and Davidson, E. H., Proc. Natl. Acad. Sci. USA 1989, 85,4710-4774. [8] Lumpkin, 0. J., Dejardin, P. and Zim, B. H., Biopolymers 1985,24, 1573-1593. [9] Slater, G. W. and Noolandi, J., Biopolymers 1989,28, 1781-1791. [lo] Deutsch, J. M., Phys. Rev. Lett. 1987, 59, 1255-1285. (111 Batoulis, J.,Pistoor,N.,Kremer, K. and Frisch,H. L., Electrophoresis 1989,IO, 442-446. [12] Stellwagen, N. C., Biopolymers 1985,24, 2243-2255. [13] Slater, G. W., Biopolymers 1988, 27, 509-524. [14] Fawcett, J. S. and Morris, C. I. 0. R., Sep. Sci. 1966, I, 9-26. [15] Kramer,O.,Greco,R. and Ferry, J.D.,J.Polym. Sci., Polym.Phys. 1975, 13, 1675-1685. [16] Lerman, L. S. and Frisch, H. L., Biopolymers 1982,21,995-997. [I71 Drossman,H.,Luckey,J.A.,Kostichka,A. J.,D’Cunha, J. and Smith, L. M., Anal. Chem. 1990, 62,900-903.

Carlo Tonani’ Michel Faupel’ Pier Giorgio Righetti’

Isoelectric membrane simulator: A computational approach for isoelectric Immobiline membranes

Sciences lDepartrnent Of and Technologies, University of Milano 2Exp10ratoryResearch and Services9 Chromatography Laboratory, Ciba Geigy Ltd., Basel

Isoelectric membrane simulator (IMS) is a computer program meant for computation of pH, buffering power (p),ionic strength (I> and dissociation degree ( a ) of a mixture of up to 3 buffering and 1 titrant Immobilines, for generating in a reproducible manner isoelectric membranes. Such membranes, of precise isoelectric point, are then used for large-scale protein purification by isoelectric focusing in multicompartment electrolyzers. IMS can be used, in a more general application, for titrating mixtures of buffers to a desired pH value. This versatile program is written in M.Q. BASIC rel. 2.5 and it runs on any IBM hardware or compatible machine supported by MS-DOS. An example of purification of superoxide dismutase in a multicompartment electrolyzer with a set of fixed p l membranes of widely differing composition is shown.

1 Introduction

based on the ‘tandem’ principle, i.e. on the use of only two Immobiline (the acrylamido weak acids and bases used to generate and maintain the pH gradient in IPG) species, one buffering and the other titrating within thedesired pH interval. However, when several buffering species had to be mixed and titrated to form an extended pH gradient, the problem became quite complex. We thus resorted to automatic computation for simulating the gradient parameters (pH, ionic strength, buffering power) and for optimizing such formulations.

Soon after publishing the fundamentals of the technique of immobilized pH gradients (IPG) [l],it became apparent to us that IPGs would not survive if we could not find a way to compute recipes for extended pH gradients. We recall here that IPGs were born as a continuation of conventional isoelectric focusing (IEF) in carrier ampholyte (CA) buffers [2] and originally were only meant to extend the fractionation capability of the conventional technique to extremely narrow pH ranges. In fact, in the cornerstone publication [l], only modified Henderson-Hasselbalch’s equations were Our first simulation program (MGS, or monoprotic electrogiven for computing narrow to ultranarrow pH gradients, lyte gradient simulation) was operating by the end of 1982. extending to a maximum of 1 pH unit. Such equations were A first approach to the formation of extended pH gradients was through the sequential elution of buffering species of increasing pK from a five-chamber mixer [3], developed Correspondence: Prof. P. G. Righetti, University of Milano, Via Celoria 2, from an earlier idea of Peterson and Sober [4].This cumberMilano 20133, Italy some procedure was soon abandoned in favor of standard two-vessel gradient mixing [5,6] for which we studied the Abbreviations:J, buffering power; %C,(g of cross linker)/%T. IEF, isoelecconditions for gradient linearity, as a function of pK distritric focusing; IMS, isoelectric membrane simulator; IPG, immobilized bution of the buffers, and of the titrants [6].We could thus pH gradients; SOD, superoxide dismutase; %T, gel concentration in % produce formulations for a series of wide immobilized PH (g/lOO mL) 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

0173-0835/91/0909-0631 $3.50+.25/0

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gradients (spanning 2-6 pH units within the pH 4-10 range), optimized in terms of gradient linearity [7]. In this paper we compared two approaches to the generation of extended pH gradients: (i) in one case each buffering Immobiline had the same concentration in both vessels of the mixer; (ii) in the other, different concentrations of buffering ions could be present in each chamber. In the case of two-dimensional maps, however, best resolution in the focusing dimension would clearly be obtained by nonlinear pH gradients, following the relative abundance of isoelectric proteins along the pH scale. Thus, we also calculated wide, nonlinear IPG recipes for use in two-dimensional maps and in most cases requiring analysis of highly heterogeneous samples [8].As at that time we had given extended recipes including also two noncommercially available components (a strong acid and a strong base) we were asked by colleagues to publish a new set of formulations, optimized in the absence of such titrants [9]. At the beginning of 1986, we started thinking of expanding the fractionation capability of IPGs: up to this moment, the most extended pH interval described was a pH 4-10. For this reason, we had not included the dissociation products of water (H' and OH-) in our simulations, since within the pH 4-10 range their concentration is negligible. At that time, we started focusing dansylated amino acids (which exhibited PIS in the pH 3-4 interval) [lo] and we realized that there was a strong divergence between calculated and experimental pH gradients: thus our computer program was expanded to include the effects of H' and OH- on /?,ionic strength and pH profile [l 11. As a provocative concept, we introduced the idea of water as two 'Immobiline' species, one with pK=-1.74 [H'], the otherwith pK= 15.75 [OH-]. In fact, simulations were not only limited to acidic, but included also quite basic (pH 10-11) intervals [12]. In the latter case, we were able to model IPG behavior by modifying the more general program for steady-state IEF, as developed by Palusinski etal. [I31and byBieretal.[14].As chemicals with more acidic and more basic pKs became available, extended formulations including pH extremes were computed: the widest pH range that could be formulated was a pH 2.5-11 interval, spanning 8.5 pH units [15]. In 1988, we started a long-range program on the characterization of existing Immobilines [16, 171 and on the synthesis of new species (a pK3.1 [18], a pK6.6 and a pK7.4 [19], a pK8.05 [20] and a pK 10.3 [21]).At this point the family of'Immobilines' had expanded considerably and our former program (which was limited to mixtures of no more than 10 species, including buffers and titrants) could not handle the new generation any longer. These factors forced us to develop a brand-new program, PGS (polyelectrolyte gradient simulation) for IEF in IPGs and for chromatography [22,231. More recently, we have extended the IPG technique in other directions: for preparative purposes, a multichamber electrolyzer with Immobiline membranes has been described [24,25]. In addition, Immobiline pearls, with specific isoelectric points, have been produced [26]. For preparation of such membranes and beads, we needed a simple program for calculating the isoelectric point (pl), together with buffering power (p),dissociation degree ( a ) and ionic strength (I) of such systems. We describe in the present paper the characteristics of this computer program, which has been modified from our previous PGS package, and has been called IMS: isoelectric membrane simulator.

2 Materials and methods 2.1 Superoxide dismutase Recombinant, human superoxide dismutase (rh-SOD), cloned and expressed in E. coli, was a gift from Dr. E. Wenisch (University of Agriculture and Forestry, Vienna, Austria). 2.2 Equipment and chemicals for electrophoresis All IPG experiments were performed in the LKB 2117 Multiphor I1 horizontal electrophoresis system coupled with the LKB 2297 Macrodrive 5 power supply and Multitemp I1 thermostat. IPG gel casting was carried out by using the LKB 2117-903 2D gradient and Immobiline gel kit. Acrylamide, N,N'-methylenebisacrylamide (Bis), N,N,WN-tetramethylethylenediamine (TEMED), Repel Silane, GelBond PAG film, Immobilines and Ampholine were purchased from Pharmacia LKB Biotechnology (Uppsala, Sweden). 2.3 Analytical IPGs

The technique is described in detail in [27]. Briefly, analytical IPG gels in the pH range 4.4-5.4 were prepared as 4%T, 4 % c matrices, containing 368 pL ofImmobiline pK4.6 and 147 WLof Immobiline pK 9.3 (for the 8 mL solution at the pH 4.4 extreme) and 604 pL of Immobiline pK4.6 and 544 pL of Immobiline pK9.3 (for the 8 mLsolution at the pH 5.4 extreme; each stock solution was 0.2 M). After casting and polymerization, the gel was washed 3 times in distilled water, dried and reswollen in 0.6% Ampholine pH 4-6.The gel was then run at l O T , 2000 V,,, for a maximum of 8-10 h, with both anodic and cathodic filter paper strips soaked in the Ampholine reswelling solution. Staining was in Coomassie Brilliant Blue R-250 in 10% acetic acid, 30 O/o ethanol in presence of 0.1% copper sulfate [2]. 2.4 Preparative runs in the multicompartment electrolyzer The electrolyzer was run with three sample chambers and two electrolyte reservoirs. Thus, 4 membranes were made for sample fractionation: a pZ4.4 at the anode, a pZ5.4 at the cathode and two central ones, for collecting the PI5.2 isoform (the most abundant and most enzymatically active; see results): a pI5.15 and a pZ5.25 membrane, with pls encompassing the p l value of the SOD-isoform of interest. These two latter membranes (containing 7%T, 4%C neutral monomers) were made in four different modes: one by interpolating the Immobiline molarities from the analytical gel (control), the other 3 by computer simulation with arbitrary Immobiline values using the IMS program. The control membranes (referred to as M,) were: a) pZ5.15: 12 mM pK4.6+9.3 mM pK9.3(/3=4.75,1=9.36);b)pZ5.25:12m~ pK 4.6 + 9.8 mM pK 9.3 (p = 4.14; I=9.8). The other three types of membranes made by computer simulations were calculated to contain, respectively, 1,2 or 3 buffering Immobilines and, in all cases, 1 titrant species. They will be referred to as M,, M, and M,, respectively. Their composition was as follows (in parentheses: a , dissociation degree; p, buffering power in mequiv. L-' pH-'; I, ionic strength in mequiv. L-'):

Isoelectric Immohiline membranes

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MI: a) pZ 5.15: 8 mM pK 6.2 ( a = 0.92) + 9.42 mM pK 4.6 ( a = 0.78) ( p = 5.12, l = 7.35); b) PI5.25: 8 mM pK 6.2 ( a = 0.90) + 8.8 mM pK 4.6 ( a = 0.82) (p = 4.7; I = 7.2); M,: a) pl5.15: 6 mM pK 6.2 ( a = 0.92) + 6 mM pK 4.6 ( a =0.78) + 0.84 mM pK3.1 ( a =0.99) (p =3.44; 1=5.5); b) pZ 5.25: 6 mM pK 6.2 ( a = 0.90) + 6 mM pK 4.6 ( a = 0.82) + 0.5 mM pK3.1 ( a = 0.99) (p = 3.33; I = 5.39); M,: a) p l 5.15: 10 mM p k 6.2 ( a = 0.92) + 5 mM pK 4.6 ( a =0.78) + 3 mM pK3.6 ( a =0.97) +2.37 mM pK1 ( a = 1.00) (p=3.9;1=9.18); b)p15.25: 1 0 m ~ p K 6 . (a=0.90)+5mM 2 pK4.6 ( a = 0.82) + 3 mM pK3.6 ( a = 0.98) + 1.98 mM pK 1 ( a = 1.00) (p = 3.97; I = 9.0). The electrolyzer was loaded with a total of 100 mg of rhSOD (desalted by gel filtration) dissolved into 75 mL (25 mL per chamber) and run overnight at 600 V (over a 5 cm electrode distance), 10“C,5 mA. Anolyte; dilute acetic acid to pH 4.2; catholyte: 50 mM gamma-aminobutyric acid, pH 6.8. At the end, only the sample contained in the central chamber (delimited by the anodic, pl5.15, and the cathodic, pZ5.25, membranes) was analyzed. The same experiment was repeated another 3 times with the couples of MI, M, and M, membranes, respectively, calculated with the IMS program as described above. The analytical gel of all four runs is shown in the result section.

633

The water contribution is also taken into account, with K,= The Immobiline concentrations are entered in millimoles (mM, with the results expressed in the same unit; the titrant higher concentration accepted by the program is 1 M). The heart of the titration’s module algorithm is a bisection procedure, slower than the Newton procedure, but always converging [22]. 3.1 The main menu

The main menu gives the following options: D)ata entry: by pressing the D key one enters the Immobiline manager, which allows selection of Immobilines species and their concentration. T)itration: by pressing the T key one can titrate to a desired pH a mixture of up to 3 buffering Immobilines. S)tart over: when pressing the S key the program resets and is ready for a new work session. H)elp: by pressing the H key one gets information on how to use IMS; P)rint: this option gives a hardcopy of the results of computation. Q)uit: this option causes the end of the program and the control is returned to DOS. 3.2 The Immobiline manager

Figure 1 gives the architecture of the IMS program. The program, developed on an AT& PC-6300, runs on PC-IBM or compatibles based on the old generation CPU 80868088 as well as on a new generation of 80286 or 80386 microprocessors. The requirements are: a RAM memory of at least 256 Kb, a floppy disk drive and MS/DOS 3.14 and successive versions. The equations implemented for calculation are in their extended version, as described in [3,5,6].

When entering the manager, and pressing the D key, an option is given for selecting up to three buffering Immobilines, which are entered by their code number. We shall give here a practical example: generation of a p16.6 membrane by using three buffering Imrnobilines: pK6.2,pK6.6 and pK 7.0, each at a concentration of 5 mM. After entering these values, the computation of Table 1 will appear on the screen: name of each species, type (acid or base), concentration, dissociation degree and M, value. This is followed by the pH of the solution (pH 9.445, since these are free bases), the buffering power value (in milliequivalents L-’ pH-’) and the ionic strength of the mixture (in milliequivalents L-’).Note that both values refer to the pH of this mixture of bases.

I S O E L E C T R I C

3.3 The titration manager

3 About the program

M E M B R A N E

S I M U L A T O R

S T R U C T U R E

DATA ENTRY FOR IMKOBILINES

TITRATION MANAGER DATA ENTRY FOR

After performing the above operations, one leaves the Immobiline and enters the titration manager. In this module, the information requested is the end titration pH (in the above example, pH 6.6, i.e. the membrane pl) and the titrant code (in this example, the acidic Immobiline pK3.1 is chosen). When entering these data, the mixture of the 3 free bases will be titrated and the results displayed in Table 2 will appear. Note that here the dissociation degree of the 3 bases is the one at the plvalue (pH 6.6).The computergenerates the requested value, i.e. the titrants’ molarity (7.503 mM) and the new values for pH (6.6), p (7.57) and ionic Table 1. Display of the data entry option after selecting a mixture of 3 Immobilines

Name COMPUTATION OF PARAMETERS OF IMMOBILINES

---T--Figure 1. Scheme of the structure of the computer program ‘isoelectric membrane simulator’ (IMS). For details, see text.

I

Type

Concentration b M )

COMPUTATION MODULE FOR TITRATION

pK 6.2 Irnmobiline pK 6.6 buffer pK 7.0 Immobiline pH: Buffering power: lonic strength:

I FINAL RESULTS

Basic 5.000 Basic 5.000 Basic 5.000 9.4450 0.1281 (mEq. L-’ PH-’)~) 0.0139 (mEq. L-’)

a) mEq., milliequivalents

Dissociation degiee

M,

0.001 0.001 0.004

184 200 194

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Table 2. Display of the final results under the titration module Name

Type

Concentration (mM)

Dissociation degree

M,

pK 6.2 Immobiline pK 6.6 buffer pK 7.0 Immobiline Titrant: pK 3.1 species PH (Po"): Buffering power: Ionic strength:

Basic Basic Basic

5.000 5.000 5.000

0.285 0.500 0.715

184 200 194

1.000

145

Acidic 7.503 6.6000 7.5739 (mEq. L-' P H - ' ) ~ ) 7.5002 (mEq. L-')

a) This pH value is the isoelectric point ( p o of the membrane b) mEq., milliequivalents.

Table 3. Buffer information as given by the Info optiona) Species Chemical name M, Physical state Chemical property

pK 1.0 titrant 2-acrylamido-2-methylpropane sulfonic acid 207 Solid Acidic

pK3.1 buffer Species 2-acrylamidoglycolic acid Chemical name 145 MI Solid Physical state Chemical property Acidic pK 3.6 Immobiline Species N-acrylo ylglycine Chemical name 129 Mr Solid Physical state Chemical property Acidic pK 4.6 Immobiline Species Chemical name 4-acrylamidobutyric acid 157 Mr Physical state Solid Chemical property Acidic Species Chemical name Mr Physical state Chemical property

pK 6.2 Immobiline 2-morpholinoethylacrylamide 184 Solid Basic

Species Chemical name Mr Physical state Chemical property

pK 6.6 buffer 2-thiomorpholinoethylacrylamide 200 Solid Basic

Species Chemical name Mr Physical state Chemical property

pK 6.85 buffer N,N'-acryloylmethylpiperazine 154 Liquid Basic

MI 200 Physical state Liquid Chemical property Basic Species Chemical name Mr Physical state Chemical property

pK 8.5 Immobiline N,N-dimethylaminoethylacrylamide 142 Liquid Basic

Species Chemical name MI Physical state Chemical property Species Chemical name MI Physical state Chemical property

pK 9.3 Immobiline N,N-dimethylaminopropylacrylamide 156 Liquid Basic pK 10.3 Immobiline N, N-diethylaminopropylacrylamide 184 Solid Basic

Species Chemical name M, Physical state Chemical property

pK > 12 titrant N, N,N-triethylaminoethylacrylamide 198 Liquid Basic

a) Note: only the 6 buffers available from Pharmacia are called by the trade name Immobiline.

strength (7.5). This procedure can be repeated for anynumber of desired membranes for use in the multicompartment electrolyzer. When the final results are displayed, there appears also an 1)nfo option, which gives information about the Immobilines (chemical name, M,, physical state, chemical property). The 14 species today available are listed in Table 3.

4 Results Figure 2 shows the results of purification of the p15.2 band of rh-SOD in the multicompartment electrolyzer, made with the four sets of isoelectric membranes (Mo, control and M, to M,, simulated with the IMS program). It can be seen that, in all cases, the same results are obtained, suggesting that, as long as the plvalues of the membranes are not altered, widely differing Immobiline mixtures can be utilized.

5 Discussion

With the introduction of the multicompartment electrolyzer exploiting isoelectric Immobiline membranes, for largescale protein purification, we felt the need for a simple and Species pK 7.0 Immobiline reliable system for calculating the p1 of such membranes. Chemical name 2-thiomorpholinoethylacrylamide Up to now, this has been done by lengthy interpolation pro200 ME cedures, consisting ofmeasuring the plof the proteins in an Physical state Solid analytical IPG gel, and then manually calculating the comChemical property Basic position of two adjacent Immobiline membranes having p l values slightly above and below the plvalues of the protein Species pK 7.4 buffer Chemical name 3-thiomorpholinopropylacrylamide to be purified, so as to keep it isoelectric within a single ves214 MI sel of the multichamber electrolyzer (for the instrument, Physical state Liquid and general instruction on how to use the method, refer to Chemical property Basic Fig. 1 and to the text in [25]).The possibility of performing such calculations with a computer program greatly simpliSpecies pK 8.05 buffer Chemical name N,N-bis (2-hydroxyethyl)-N'acryloyl-1,3-diamino- fies the use of the technique. In fact, some general considerations can be drawn from our experience. propane

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Figuie 2. Analysis of the preparative rhSOD fractionation in the multicompartment electrolyzer with Immobiline membranes.Thegelwasan IPGpH4.4-5.4gradient in a 4%T,4%C matrix. Focusingwas for8 h at2000V, 10°C.StainingwithCoomassie Brilliant Blue R-250 in Cu". Sample: cathodic application (20 pL with a total of 20 pg for the purified sample, 40-50 pg for the controls). (l), (3), (6) and (8): control preparations of rh-SOD (from different culture broths).M,,M,,M* andM3: pl5.2 band purified in the multicompartment electrolyzer between two membranes havingaplof5.15 (anodic) andp15.25 (cathodic) and with the Immobiline compositions as described in Section 2.4.

5.1 Exchangeability of recipes

With our collected experience of computer simulations for generating extended IPG recipes, we have become aware that it is possible to obtain the same results with widely differing recipes (i.e. containing different Immobiline species with different molarities). This is in fact exemplified by the results of Fig. 2: even though four different Immobiline recipes have been used four the membranes keeping isoelectric the pl5.2 SOD isoform, the purification results have been identical. This is not unexpected, since the target function (an exact plvalue of the membrane) is respected in all the formulations. In fact the two membranes facing the chamber where the SOD isoform is kept isoelectric function like pH-stat units and thus perform with the same efficiency as long as the plvalue is not altered when using different recipes (and the buffering power kept at reasonable levels).

5.2 Molarity limit in the membranes The second question to be addressed is the upper limit in total Immobiline molarity which can be tolerated in a single membrane without altering its plvalue. It should be noted that in all our computer simulations, activity coefficients of the different Immobilines have never been introduced. In fact, while such corrections could be introduced in free solution, as a function of total ionic strength, it is not known what corrections should be introduced when these species are grafted into a polymer. Such corrections (which in practice would be reflected into changes in pK values) would most probably depend on the spatial distribution of the ionizable species along the backbone of the neutral polymer (the polyacrylamide skeleton). On the basis of the limit law of Debye-Huckel, it is in general accepted that the activity coefficient can be neglected (it is = unity) up to 10 mM ion concentration. Thus, in all our analytical IPG gels, no corrections have ever been introduced. While we have no direct experimental data to set an upper limit to Immobiline molarity in a gel (or membrane) for proper behavior, there is some indirect evidence based on membrane p l derivation through measurements of electrosmotic flow [28]. As shown by Wenger etal. [28], correct p l measurements can be performed with Immobiline concentrations (in a lO%T, 3%Cmatrix) up to 40 mM: above this value, the membrane behavior becomes quite erratic. Thus, we set a

safe limit for membrane preparation at ca. 20 mM Immobiline. This gives plenty of buffering power for a good performance of the multicompartment electrolyzer. 5.3 General use for titration experiments By inserting (or substituting) in the Immobiline manager the pKvalues of common buffers and titrants, it can be safely assumed that our IMS program could be effectively used for predicting the amount of titrant to be added in a titration experiment for reaching a desired pH value. This is particularly useful in, e.g., spectrophotometric titrations, where pH changes are usually obained by blind addition of a given amount of concentrated acid (or base) to the solution under titration. Clearly, regular pH changes can be obtained simply by using our IMS program and feeding in the molarity (and pKvalue) of the buffer in use and the target pH value. IMS will perform the titration and compute the mM of titrant required. The process can be reiterated until full titration data have been collected. Supported by a grantfrom Agenzia Spaziale Italiana (ASI)f o r protein purification and subsequent crystallization in microgravity. Received March 12. 1991

6 References [I] Bjellqvist, B., Ek, K., Righetti, P. G.,Gianazza, E., Gorg, A., Westermeier, R. and Postel,W., J. Biochem. Biophys. Methods 1982,6,317339. [2] Righetti, P. G., Isoelectric Focusing: Theom Methodology and Applications, Elsevier, Amsterdam 1983. [3] Dossi, G., Celentano, F.,Gianazza, E. and Righetti, P.G., J. Biochem. Biophys. Methods 1983, 7, 123-142. [4] Peterson, E.A. and Sober, H. A., Anal. Chem. 1959,31,857-862. [5] Gianazza, E.,Dossi, G., Celentano, F. and Righetti,P. G . ,J. Biochem. Biophys. Methods 1983, 8, 109-133. [6] Celentano, F., Gianazza, E., Dossi, G. and Righetti, P. G., Chemometr. Intel. Lab. Systems 1987, I , 349-358. [7] Gianazza, E., Celentano, F., Dossi, G., Bjellqvist, B. and Righetti, P. G., Electrophoresis 1984, 5, 88-97. [8] Gianazza, E., Giacon, P., Sahlin, B. and Righetti, P. G., Electrophoresis 1985, 6, 53-56. [9] Gianazza, E., Astrua-Testori, S. and Righetti, P. G., Electrophoresis 1985, 6, 113-117.

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[lo] Bianchi-Bosisio,A.,Righetti,P. G.,Egen, N. B., and Bier,M., Electrophoresis 1986, 7, 128-133. [11] Righetti, P. G., Gianazza, E. and Celentano, F., J. Chromatogr. 1986, 356,9-14. [I21 Mosher, R. A., Bier, M. and Righetti, P. G., Electrophoresis 1986, 7, 59-66. [I31 Palusinski, 0. A,, Allgyer, T. T., Mosher, R. A,, Bier, M. and Saville, D., Biophys. Chem. 1981,13, 193-203. [14] Bier, M., Mosher, R. A. and Palusinski, 0. A,, J. Chromatogr. 1981, 211,313-323. [15] Gianazza, E., Celentano, F., Magenes, S., Ettori, C. and Righetti, P. G., Electrophoresis 1989, 10, 806-808. [16] Chiari,M.,Casale,E., Santaniello,E. and Righetti,P.G.,Appl. Theor. Electrophoresis 1989, 1, 99-102. [I71 Chiari, M., Casale,E., Santaniello,E. and Righetti, P. G., Appl. Theor. Electrophoresis 1989, I , 103-107. [ 181 Righetti,P. G.,Chiari,M.,Sinha,P. K. and Santaniello,E., J.Biochem. Biophys. Methods 1988, 16, 185-192. 1191 Chiari,M.,Righetti,P. G.,Ferraboschi,P., Jain,T. and Shorr,R.,Electrophoresis 1990, I!, 617-620.

[20] Chiari, M., Pagani, L., Righetti, P. G., Jain, T., Shorr, R. and Rabilloud, T., J. Biochem. Biophys. Methods 1990,21, 165-172. [21] Gelfi, C . , Bossi, M. L., Bjellqvist, B. and Righetti, P. G., J. Bioehem. Biophys. Methods 1987, 15,41-48. [22] Celentano, F. C., Tonani, C., Fazio, M., Gianazza, E. and Righetti, P. G., J. Biochem. Biophys. Methods 1988,16, 109-128. [23] Righetti, P. G., Fazio, M., Tonani, C., Gianazza, E. and Celentano, F. C., J. Biochem. Biophys. Methods 1988, 16, 129-140. [24] Righetti, P. G., Wenisch, E. and Faupel, M., J. Chromatogr. 1989,475, 293-309. [25] Righetti, P. G., Wenisch, E., Jungbauer, A,, Katinger, H. and Faupel, M., J. Chromatogr. 1990, 500, 681-696. [26] Righetti,P.G.,Chiari,M. and Crippa,L.,J. Biotechnol. 1991,17,169176. [27] Righetti, P. G., Immobilized p H Gradients: Theory and Methodologv, Elsevier, Amsterdam 1990. [28] Wenger, P., de Zuanni, M., Javet, P., Gelfi, C. and Righetti, P. G., J. Biochem. Biophys. Methods 1987, 14,29-43.

Kyung-Bok Lee' Yeong-Shik KimZ Robert J. Linhardt'

Capillary zone electrophoresis for the quantitation of oligosaccharides formed through the action of chitinase

'Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 'Natural Products Research Institute, Seoul National University, Seoul

Capillary zone electrophoresis with fluorescence detection was used to analyze the products formed by chitinase acting on N-acetylchitooligosaccharide-fluorescent conjugates. Six oligosaccharides of the structure [N-acetylglucosamine( 1-4)], (where n = 1-6) were conjugated to 7-amino-1,3-naphthalene disulfonic acid by reductive amination. Each oligosaccharide-fluorescent conjugate was purified by preparative gradient polyacrylamide gel electrophoresis, semi-dry electrotransfer to a positively-charged nylon membrane and recovered by washing the membrane with salt solution. The products formed by treating each oligosaccharide-fluorescent conjugate with chitinase were analyzed by capillary zone electrophoresis. The chitinase treatment hexasaccharide-fluorescent conjugate was also examined kinetically to study the action pattern of this enzyme.

terns [2,4,5]. Chitinase can also catalyze transglycosylation reactions [9]. Previous workers relied on conventional Recently, we demonstrated the utility of gradient polyacryl- methods of analysis to study chitinases. These include the amide gel electrophoresis (PAGE) for analyzing fluores- use of radiolabeled chitin [lo] or chromogenic substracent conjugates of neutral, linear oligosaccharides before tes such as p-nitrophenyl-P-D-N,N-diacetylchitobiose [S]. and after treatment with exoglycosidases and endoglycosi- Analysis by paper chromatography [ 101, thin layer chromadases [l].Chitinase is a glycosidase, obtained from animal tography (TLC) [5] and high performance liquid chromato[2,3], plant [4,5] and microbial sources [5,6,7], which acts graphy (HPLC) with ultraviolet (UV) detection [2, 4, 61 on chitin through a unique and complex mechanism. Chi- have been used to study the mechanism and substrate spetinase hydrolyzes chitin, a water insoluble polymer of 1-4 cificity of this enzyme. In our earlier study [l]we demonlinked 2-acetamido-2-deoxy-~-glucose (GlcNAc) [8]. It can strated the high sensitivity of fluorescence detection of exhibit endo-type, exo-type, and random-type action pat- sugar conjugates but also the limitations of gel electrophoresis. These limitations are primarily difficulties in sample quantitation and in the automation of gel-based Correspondence: Dr. Robert J. Linhardt, Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa analyses. High voltage capillary electrophoresis is rapidly City, IA 52242, USA becoming an indispensable tool for the separation and microanalysis of biopolymers including peptides, proteins, Abbreviations: AGA, amido-G-acid or 7-amino-1,3-naphthalenedisul- nucleotides, and nucleic acids [ll]. The utility of capillary fonic acid; CZE, capillary zone electrophoresis; GlcNAc, 2-acetamido&deoxy-D-glucose; [GlcNAc(l+4)1,-AGA, N-acetylchitooligosaccharide- zone electrophoresis (CZE) to quantitatively analyze sulfated carbohydrates such as chondroitin sulfate and dermaAGA; HPLC, high performance liquid chromatography; PAGE, polytan sulfate derived disaccharides has recently been deacrylamide gel electrophoresis

1 Introduction

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