338

J. Pospichal e t a / .

Jan Pospichal' Dietmar Tietz' T. Roy Ittyerah' David Halpern' Andreas Chrambach' 'Section on Macromolecular Analysis, Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 2Polysciences Inc., Warrington, PA

Electrophoresis 1991, 12, 338-341

Gel electrophoresis of polystyrene particles in glutaraldehyde crosslinked polyvinyl alcohol Polystyrene sulfate and carboxylate particles (19-189 nm radius) were subjected to electrophoresis in glutaraldehyde crosslinked polyvinyl alcohol of molecular weight 25.000 and 650.000 Da at various concentrations. The degree of crosslinking is severely limited by the mechanical properties of the gels that deteriorate beyond a glutaraldehyde concentration which decreases with increasing polyvinyl alcohol chain length. The effective fiber radius of the short-chain and longchain polymer fiber was 45 f25 and 131 2 47 nm, respectively. Thus, these media do not significantly exceed the apparent fiber thicknes of agarose, are more difficult to prepare - but are well-defined synthetic products rather than natural ones, and have the advantage of carrying no net charge and can therefore be expected to exhibit no electroendosmosis.

1 Introduction

2 Materials and methods

There appears to be a correspondence between the effective gel fiber radius and the radius of the sieved particle in polyacrylamide and agarose gel electrophoresis (Table 1). Based on the hypothesis of such correspondence, an attempt was made in this study to generate polymer fibers with significantly increased effective fiber radii for the purpose of extending the size range of molecules that can be separated on the basis of size differences in electrophoresis carried out in polymer networks, i. e. by molecular sieving. Although the separation of large, Mb-sized chromosomal DNA is the ultimate purpose of the study, it was carried out with spherical standard-sized polystyrene particles since they are commercially available and are known to be amenable to size separation in gels [l-31, without recourse to pulsing of the electric field (unlike large DNA [4, 51).

2.1 Materials

Polyvinyl alcohol (PVA) was selected because (i) it carries no functional groups other than hydroxy residues[6,7] suitable for the polar interactions with hydrophilic molecules such as DNA; (ii) the absence of charged groups eliminates electroendosmotic back-flow; (iii) the polymer fibers are commercially available in a large selection of chain lengths (and therefore need not be polymerized in the laboratory), and are stable to storage; (iv) it is chemically stable at any pH. Glutaraldehyde crosslinking [8] was used in this first approach toward making thick polymer fibers because it required no prior derivatization of PVA with an activating group, such as the reaction of PVA with acryloyl-chloride or epoxy-groups [9]. PVA at a high concentration (10%) in 40 O/o glycerol forms a gel at 0 "C, which, in electrophoresis, exhibits an effective pore size similar to that of polyacrylamide gels within a conventional %T and %C range [lo]. PVA-polyethylene glycol and PVA-borate gels were investigated byRighetti and Snyder [ l l ] but have not as yet been evaluated with regard to their sieving properties. Correspondence: Author: Dr. A. Chrambach, Bldg. 10, Rm. 6C101, NIH, Bethesda M D 20892, USA Abbreviations: %C, glutaraldehyde (v/v from a 25 O/O stock solution); CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]- l-propanesulfonate; HEM, N-b-hydroxyethylmorpholine; Nycodenz, 5-(N-2,3-dihydroxypropylacetamido)-2,4,6-triiodo-N,N'-bis(2,3 dihydroxypropy1)isophthalamide; PVA, polyvinyl alcohol; SPADNS, 2-(p-sulfophenylazo)1,8-dihydroxy-3,6-naphthalenedisulfonicacid, trisodium salt; VoT, PVA concentration (w/v)

0VCH Verlagsgesellschaft mbH, D-6940 Weinheim,

1991

PVA ( M , 650 000, Grade "Ultra High", 99.5 O/o hydrolyzed), a sample of an experimental product, and PVA ( M , 25000, 98.5% hydrolyzed,Cat.No. 4397,Batch 85609) were obtained from Polysciences (Warrington PA 18976-2590). PVA ( M , 124 000-186 000, 98-98.8% hydrolyzed) was Airvol 350 (Air Products and Chemicals Inc., Allentown PA). Polystyrene size standards were those defined by Table 2. Glutaraldehyde (25 Yo solution, Aldrich Chem. Co., Milwaukee WI 53233, Cat. No. G-400-4, Lot 01325BM), 3-[(3-cholamidopropyl)-dimethylammonio]l-propanesulfonate (CHAPS, Calbiochem-Behring, La Jolla CA 92037, Cat. No. 220201), 2-(p-sulfophenylazo)-1,8-dihydroxy-3,6naphthalene-disulfonic acid (SPADNS, Aldrich Chem. Co., Milwaukee WI 53233, Cat. No. 11, 475-8), N-@-hydroxyethylmorpholine (HEM, Fluka Chemie, Buchs, Switzerland,Cat.No. 54409,Analysis No. 288162 189) and 5-(N-2,3dihydroxypropylacetamido) -2,4,6 - triiodo - N,M - bis(2,3 dihydroxypropy1)isophthalamide (Nycodenz), Nycomed, Oslo, Norway, Batch No. 9063978, obtained from Accurate Chemicals and Scientific Corp., Westbury NY, Cat. No. AN7050) were used.

2.2 Preparation of crosslinked PVA gels for electrophoresis Stock solutions of PVA-650 and PVA-25 were prepared at 4 and 10% w/v in water at 90°C by stirring for 5 h or until lumps had disappeared completely upon subsequent ultrasonic degassing (Ney Ultrasonik, Yucaipa CA, Model 20/ H). Stock solutions were diluted to give a final concentration of 0.1 N HCl, 10% Nycodenz, 10-20 mM CHAPS. The mixture was stirred and glutaraldehyde was added to a concentration 0f0.007~/0forPVA-650 and 0.1% forPVA-25.The mixture was degassed ultrasonically for 2 min. Two mL aliquots were dispensed into 6 mm ID, 8 mm OD quartz tubes, sealed at one end with a dialysis membrane held in place by a sleeve (section of Tygon tubing), and positioned in a test tube rack. Tube contents were overlayered with 0.01 O/o aqueous SPADNS to a height ofabout 3 mm and the tubes were heated to 50" C for 30 min, allowed to cool at room temperature for about 30 min, and then preelectrophoresed at 10 mA/tube for 2 h, 25 "C, in a gel tube apparatus of rectangular geometry [12]. The operative gel buffer, i. e. its composition behind the MES-/Cl- moving bound0173-083519 1/0505-0338 $3.50+.25/0

Electrophoresis in crosslinked polyvinyl alcohol

Electruphoresis 1991, 12, 338-341

catholyte, as well as SPADNS to a pink color. Electrophoresis was allowed to proceed at a regulated potential of 1 V/ cm. Gels were photographed by Polaroid camera under direct light against a black background. Relative mobilities ( R J were calculated as the migration distance of the zone divided by the displacement of the chloride-MES boundary marked by SPADNS.

ary, was 0.1120 M 2-(N-morpholino)ethanesulfonic acid (MES),0.2427 M HEM,pH7.04,0.10 M ionicstrength,25”C. The prepared gel buffer consisted of 0.0949 M HEM, 0.0557 M HCl (final concentration), pH 6.74. The catholyte was 0.06 M MES, 0.03 M KOH, pH 6.12.The anolyte was 0.06 M HEM, 0.03 M HCl, pH 6.89. Since each tube contained SPADNS from the overlayering solution, the endpoint of pre-electrophoresis could be recognized as the attainment of L2 of the tube length by the dye*.When HEM’, replacing H’ in the tube, reached the zone of SPADNS, the dye accelerated and started to diffuse due to increased Joule heating. The catholyte reservoir was emptied of buffer and was then filled with water. The contents of the tubes above the PVA column were carefully withdrawn into a syringe. The reservoirwas then filled with K-MES catholyte and the contents of the tubes above the PVA column were again withdrawn by syringe.

2.4 Data processing A Macintosh I1 personal computer was used. &values were entered into data files in the format shown in Fig. 5 of [13]. Program ELPHOFIT [13] computes the fiber radius, r(nm), directly from Eqs. (1) and (2) of [14]. Standard errors given (+ in Table 3) are the standard deviations of parameter estimates and assume a Gaussian distribution.The rootmean-square (RMS) error is a dimensional measure of the goodness of fit. It states an “average” deviation of a data point from the value predicted by the model. “Perfect fit” gives an RMS error of 0. The determination coefficient is another measure of the relative goodness of fit. It states the

2.3 Electrophoresis

The surface of the PVA column remained covered by a 0.5 mni layer containing Nycodenz. The sample was applied onto the sharp surface of that layer to avoid the turbulence and floating of the sample that was observed when it was applied directly onto the PVA column. The sample of 4 polystyrene particles was made 25-50 pL of a solution containing a 2 : 1 : 1 (v/v/v) mixture of 4 polystyrene latex suspensions (Table 2), 100mM CHAPS and 4 X concentrated

*

Table 3. Gel fiber properties of crosslinked PVAdl PVA (kDa)

~

The transference numbers ofH+ and CI.relate as 0.82/0.18=4.5.The ascending HEM+ therefore meets the descending C1- at U4.5 of the length of the plolymer column. SPADNS has about one half the mobil. ity of Cl-. The continuation of pre-electrophoresis to 1/2 of the column length therefore incorporates a safety margin by a factor of approxima tely 5.

Fiber volume (mL/g dry matrix)

RMS error x 102

130.8

67.7

8.26

k 47.2

f 159

Fiber radius (nm)

25

44.7

20.5

f 25.4

f 17.6

Particle radius (nm)

Gel

Effective fiber radius (nm)

Reference

Protein

1 to 6

1 to 3

[151

Protein

1 to 4

Polyacrylamide 1 to 5 % C ~ i , Agarose 0.4 to 1.6% 0.4 to 8 % 2 to 10 “/o Polyacrylarnide 10, 20%C&, Agarose 0.4 to 1.6 0.4 to 1.6 0.4 to 3 % Agarose

Viruses

13 to 31

Polystyrene

15 to 43

0 to 2 5 to 10 1

3, 1 21 to 54 20 to 25 11 to 36 21,36

Table 2. Polystyrene latex size standards No.

Radius (nm)

Charged group

Manufacturer

Cat. No.

Vo Solids

1 2 3 4 5

19 30.5 47 103 189

Sulfate Carboxylate Carboxylate Carboxylate Sulfate

IDC‘” Pol ysciences Polysciences Polysciences IDCa’

2-90-24 19773 16662 19391 10-15-50

8.6 2.5 2.5 2.5 86

a ) IDC, Interfacial Dynamics Corp., Portland OR 97207

12.1

0.977 0.839

a) For definitions of the statistical parameters see Section 2.4.

Particle

1 to 4

Determination coefficient

~~

650

Table 1. Commensurate values of effective fiber radius and particle r d i u i

Protein

339

340

J. Pospichal e t a / .

Elecrrophorcsis 1991, 12, 338-341

fraction of the total sum of squares corrected for the mean that is accounted for by fitting the model to the data. “Perfect fit” corresponds to a value of 1. The computer output summarized by Table 3, specifying the effective fiber radius, among other parameters, was obtained in the format of Fig. 7 of [13]. In the case of PVA-2.5, the upper limit of the constraints was increased to 10” since the Ferguson plots covering an extremelynarrow O/oT range are steep and linearly extrapolate to extremely high Yo values.

Table 4. Gelation times (h) of 0.025 O/o glutaraldehyde crosslinked 3% PVA-(124-186) at room temperature as a function of acidity

3 Results

3.2 Determination of the effective fiber radius of glutaraldehyde crosslinked PVA-650 and PVA-25

3.1 Limitations in the degree of crosslinking by glutaraldehyde of PVA The degree of crosslinking of PVA by glutaraldehyde cannot be increased beyond the limits set by the mechanical gel properties (Fig. 1). Beyond that limit of glutaraldehyde concentration, gels became brittle, cloudy, synerese, and separate from the glass walls. The maximal glutaraldehyde concentrations within these constraints appear to be the same for PVA-650 and PVA-( 124-186). However, much higher degrees of glutaraldehyde crosslinking still provide stable, clear gels with PVA-2.5.Gelation is accelerated in pro-

HC1 (N): Time (h):

0.01 11.5

0.02 9.5

0.05 2.8

0.10 1.2

0.20 0.5

0.40 0.25

portion to the degree of acidity (Table 4). Gelation at room temperature overnight provides an effective pore size somewhat smaller than that obtained after 1 h at 50°C.

Polystyrene particles ranging in molecular radius from 19 to 189 (Table 2) were subjected to gel electrophoresis (1 V/cm; 10mMCHAPS; .5-10% Nycodenz; 25°C; 0.1 M ionic strength) at pH 6.74 in the discontinuous buffer system detailed above. A representative gel pattern and Ferguson plots for PVA-650 are shown in Fig. 2.

0.14 0.12

Iv

h

8

Y

g z

0.10

I

5a

0.08

LT

2 3 c7

0.06

1 0.8

0.04

0.6 Y-

U 0.02

0.4

0.2

0

0

1

2

3 4 PVA (Yo)

5

Figure I . Properties of glutaraldehyde-crosslinked PVA as a I’unction of the concentrations of PVA and glutaraldehyde: PVA-(124-186) was reacted with glutaraldehyde at room temperature overnight in 0.3 N HCI; PVA-25 and PVA-650 were reacted at room temperature overnight in 0.1 N HCI. Each size of PVA gives rise to crosslinked polymer with 4 classes of physical properties, designated I to IV in the figure, as a function of the concentration of glutaraldehyde used.As the crosslinker concentration is raised, the mechanical properties of each size of PVA tested deteriorates. This deterioration of gel quality sets an upper limit of crosslinking for the production of crosslinked PVA gels useful in electrophoresis.

0.1

%T Figure 2. Ferguson plots ofpolystyrenc bulfate particles in 0.007% glutaraldehyde crosslinked PVA-650: electrophoresis of 25 pL samples in MESH EM buffer (see Section 2.3), pH 6.74, 0.10 M ionic strength, 10 mM CHAPS, 10% Nycodenz, with SPADNS as a tracking dye,25’C.The pen markings within bands designate the visually estimated concentration maxima used to measure migration distances.

Elecliophoresis 1991, 12, 338-341

Electrophoresis in crosslinked polyvinyl alcohol

The relation between mobility and gel concentration depicted as the Ferguson plot (Fig. 2B), mathematically stated as

can be interpreted in terms of the fiber radius, r, since its slope, K,, is given by

KR= 0.01 X VF/? X ( r + R)’ The radii, R, of standard-sized particles are known; K, is obtained from the measured Rfvalues as the slope of the Ferguson plots of each particle, and the fiber properties r and the fiber volume VF (mL/g) are obtained by simultaneously fitting Eqs. (1) and (2) to R, and O/oT [ 13,141.The effective fiber radii of 131 and 45 nm for PVA-650 and PVA-25, respectively (Table 3), show that representative, feasible degrees of glutaraldehyde crosslinking ofboth long-chain and short-chain PVA produce fiber thicknesses not significantly larger than those obtainable with agarose at low gel concentrations [IS, 161. PVA-25, crosslinked with 0.1 O/o glutaraldehyde, in the concentration range of 1.8-2.00/0, allows for the gel electrophoresis of polystyrene carboxylate with R= 189 nm, and yields the convex Ferguson plot shown in Fig. 3.

341

Instead, we attempted to answer the question whether, by crosslinking ofPVA chains, one can generate gel fibers with significantly larger effective fiber radii (as defined previously [15]) than is possible with the conventional sieving medium for large DNA agarose. This effective fiber radius is 10-36 nm in the agarose gel concentration range of 0.43 O/o [ 161.The effective PVA fiber radii of 131 and 45 nm for PVA of M, 650 000 and 2.5 000, respectively, are not by at least one order of magnitude greater - i. e. “significantly” greater for the purposes of this investigation -than those found for agarose. Glutaraldehyde crosslinked PVA, at least of the types tested, therefore does not provide a medium to test the proposition whether particles of 1 p or more in radius can be sieved in commensurate fiber networks. The glutaraldehyde crosslinked PVA gels generated in this study allow for the gel electrophoresis of a polystyrene carboxylate particle with 189 nm radius. This is slightly more than the 122-138 nm polystyrene but less than the SO0 X (1000-3000) nm dimensions of an E. coli particle reported to be amenable to agarose gel electrophoresis [17]. However, the absence of electroendosmotic flow may be an advantage in some separations, as is also the mechanical stability of the gels compared to very dilute agarose. Received November 6, 1990

I

I

I

0-

- 0 29

-

-02

n

--

I

I]

- 0.07

U,

-3-

- 0.03

\ -1.6

I

5 References

- 0.43

-. -.4 -

5

- 00 705 65 54 -

I

1.8

I

1.85

I

I

1.9

I

I

1.95

I

I

2

%T

Figure 3. Ferguson plot of polystyrene sulfate with a molecular r‘idius of 189 nm in crosslinked PVA-25: Conditions as in Fig. 2.

4 Discussion The choice of glutaraldehyde as the crosslinking agent necessitates gelation in acidic solution, and the subsequent electrophoretic exchange of the acid for buffer is time consuming. Moreover, it is not known whether this exchange is sufficiently quantitative to eliminate the possibility of a reaction of residual glutaraldehyde with the electrophoresed particle. Making PVA into a useful electrophoresis medium, possibly by application of other crosslinking agents reactive with the OH-groups of PVA in preference to those of water, or by coupling it to copolymers [ll], was not the purpose of this study.

[ l ] Gomhocz,E.,Tietz, D., Hurtt, S. S. and Chramhach,A.,ElectrophoreS ~ S1987, 8, 261-271. [2] Hahn, E., Wurts, L., Tietz, D. and Chrambach, A,, Electrophoresis 1988, 9,243-255. [3] Righetti, P. G., Brost, B. C. W. and Snyder, R. S., J. Biochem. Biophys. Methods 1981,4,347-363. [4] Carle, G . F., Frank, M. and Olson, M. V., Science 1986,232, 65-68. [5] Gemmill, B. J., Adv. Electrophoresis 1990, 4, 1-48. [6] Pritchard, J. G., Poly(Vinyl Alcohol), Gordon and Breach Science Publ., London 1970, Chapter IV, pp. 57-80. [71 Finch, C. A.,Polyvinyl Alcohol, J. Wiley, London 1973, pp. 1-622. [8] Higuchi, A. and Iijima,T., Polymer 1985, 26, 1207-1211. [9] Sundberg, L. and Porath, J., J. Chromarogr. 1974, 90, 87-98. [lo] Reich, G. and Sieber, H., Z. Chem. 1966, 6,351-352. [ l l ] Righetti, P. G. and Snyder, R. S., A p p ( . Theor. EleCtrophoreSiS 1988, 1, 53-58. [12] Fawcett, J. S. and Chrambach, A. Electrophoresis 1986, 7, 260-265. [I31 Tietz, D., Electrophoresis 1990, 12, 28-38. [14] Tietz, D., Aldroubi, A,, Schneerson, R., Unser, M. and Chrambach, A., Electrophoresis 1990, 12, 46-54. [15] Rodbard, D. and Chrambach, A , , Proc. Nuti. Acad. Sci. USA 1970.65, 970-977. [16] Tietz, D. and Chrambach, A., Anal. Biochem. 1987, 161,395-41 1. [I71 Serwer, P., Moreno, E. T. and Griess, G . A,, in: Schafer-Nielsen, C. (Ed.) Electrophoresis ’88,VCH Verlagsgesellschaft, Weinheim 1988, pp. 216-222. [18] Buzas, Z. and Chrambach, A,, Elecfrophoresis 1982, 3, 130-134. [19] Rodbard, D., Levitov, C. and Chrambach, A,, Separation Sci. 1972, 7, 705-723. [20] Tietz, D. and Chrambach, A., Electrophoresis 1986, 7, 241-250. [21] Serwer, P., Allen, J. L. and Hayes, S. J., Electrophoresis 1983,4,232235.