Size exclusion chromatography and size exclusion HPLC of proteins

149 SUB mapillust C O L O R 7: x$ = C H R $ ( 1 3 4 ) L O C A T E 23, 22: P R I N T "pH": L O C A T E 2, I: P R I N T '~-Lo~'; M e $ L O C A T E 2, 41: P R I N T " F R E E E N E R G Y M A P F O R A T P H Y D R O L Y S I S " L O C A T E 3, 46: P R I N T " ( p H v s "; Me$; ")" L O C A T E 5, 43: P R I N T " - 8 . 2 k c a l < n; : C O L O R 0: P R I N T x$: C O L O R 7 L O C A T E 6, 43: P R I N T " - 8 . 2 - 8.4 "; : C O L O R I: P R I N T x$: C O L O R 7 L O C A T E 7, 43: P R I N T " - 8 . 4 - 8.6 "; : C O L O R 4: P R I N T x$: C O L O R 7 L O C A T E 8, 43: P R I N T " - 8 . 6 - 8.8 "; : C O L O R 5: P R I N T X$: C O L O R 7 L O C A T E 9, 43: P R I N T " - 8 . 8 - 9.0 "; : C O L O R 2: P R I N T x$: C O L O R 7 L O C A T E 10, 43: P R I N T " - 9 . 0 - 10 "; : C O L O R 3: P R I N T x$: C O L O R 7 L O C A T E 11, 43: P R I N T "-10 - 11 "; : C O L O R 6: P R I N T x$: C O L O R 7 L O C A T E 12, 43: P R I N T "-11 > "; : C O L O R 7: P R I N T x$: C O L O R 7 F O R i = 46 T O 322 S T E P 46 L I N E (51, i ) - ( 5 6 , i): L I N E (300, i ) - ( 3 0 5 , i) L O C A T E i / 16 + .5, 5: P R I N T U S I N G "@"; i / 46 NEXT F O R i = 0 T O 244 S T E P 48 L I N E (i + 56, 0 ) - ( i + 56, 4): L I N E (i + 56, 3 3 2 ) - ( i + 56, 336) L O C A T E 22, 8.5 + i / 8: P R I N T U S I N G "#"; i / 48 + 4 NEXT END SUB

Size E x c l u s i o n C h r o m a t o g r a p h y and Size Exclusion H P L C o f Proteins S A A D T A Y Y A B , S E E M A Q A M A R and MOZAFFARUL ISLAM

Interdisciplinary Biotechnology Unit Aligarh Muslim University Aligarh 202002, UP India Introduction Size exclusion chromatography (SEC) is also known as gel filtration, gel permeation or molecular sieve chromatography. A number of articles on gel filtration of proteins have appeared 1-4 but none of them dealt with all aspects of SEC. In this paper we describe the methodology and applications of SEC including size exclusion HPLC ( S E - H P L C ) with the aim of familiarizing people how to use this powerful technique without the need for sophisticated instrumentation. SEC may be used to fractionate and characterize proteins according to size. The sieving medium is a porous gel. Molecules much smaller than the pore diameter will have more probability of penetrating the gel and will pass through the column more slowly. The actual speed of movement of each component in a mixture is dependent on the ease with which molecules can pass into the gels and be retarded. Molecules with diameter much larger than the pore size will have less probability to penetrate the gel particles and will be excluded from the gel and will pass through the column unimpeded. Intermediate size molecules can pass into some of the gel particles but compared to very small molecules, a greater proportion of the intermediate size molecules will be outside the gel at any time. The most widely used types of the gels are cross-linked dextrans (Sephadex), crosslinked agarose (Sepharose), cross-linked polyacrylamide (Biogel), cross-linked allyldextran (Sephacryl) and controlled pore glass beads. These are graded according to pore size making it possible to vary the range of molecular weight which can be fractionated. The upper limit of the fractionation range is the exclusion limit which means that the molecules with molecular weight greater than this will have less probability of penetrating the gel and will be completely excluded. SEC may be carried out on a large scale but since large columns are rather time-consuming to run and the gel media required to fill them expensive, the method finds most application in the later stages of protein purification. Packing the column Dry gels are allowed to swell in water or buffer under conditions specified by the manufacturer. If gel is preswollen, then it is directly processed for the removal of fines which is important because the presence of fines can block the column. Fines are BIOCHEMICAL

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removed by repeated decantation. A glass column of uniform diameter previously washed with chromic acid, detergent and water is mounted in a vertical and vibration-free position. The outlet of the column is connected to tubing with a screw stopcock for regulation of the flow rate. The diameter of the column may be determined at several places along the height of the column by collecting a known amount of water in to preweighed weighing bottles whose volume can be taken as the volume of a cylinder. A small amount of glass wool previously boiled in water is placed at the bottom of the column and its surface is covered with few glass beads. After filling one-third volume of the column with the operating buffer, degassed gel slurry is poured slowly into the column with the help of a glass rod. The column should be packed in a single step to avoid discrimination among the beads during settling and an extension should be used if necessary. The gel is left to settle under gravity overnight. When the gel has formed a smooth upper surface, the outlet is opened with a flow rate of 5 mi/h. As the gel settles down, the flow rate is increased gradually. The gel bed should be stabilised by passing three bed volumes of eluent.

Experimental determination of chromatography parameters The three parameters Ve, Vo and Vi are used to describe the behaviour of a molecule on a gel filtration column and these must be determined experimentally. The elution volume (Ve) is the volume of eluent collected from the start of loading the sample to the point of its maximal elution. The behaviour of a solute is described by its Kd (distribution coefficient) value which is the fraction of inner volume (Vi) accessible to a solute molecule: K d = (V e - Vo)/Vi Value of K d will be zero for solutes totally excluded from the column and 1.0 for solutes to which the solvent both within the pores and in the void volume is equally accessible. A Kd >1.0 indicates adsorption or ionic interactions between solute and the gel material. It is difficult to determine the exact value of V~ since some inner volume is occupied by the gel matrix (Vm) and bound water to it. Therefore it is usual to take the available value of Ka, ie Kav:

~:~ = (vo - V o ) / ( v , - Vo) The void volume (Vo) is the volume of interstitial liquid. Molecules with diameter larger than pore size are completely excluded from the gel and their elution volume is equal to void volume of the column. Blue dextran (a dextran with a blue dye chemically linked to it, Mr 2 x 106) is completely excluded from Sephadex, polyacrylamide gels and some agarose gels and may be estimated by extinction measurements at 625 nm and is widely used for the determination of the void volume. Caution is necessary when blue dextran is mixed with proteins, however, since it forms complexes with some proteins. With spherical beads, Vo is 30-35% of the total volume (Vt) depending on how tightly the column is packed. Thus the useful range for resolving proteins lies within about 80% of the remaining volume (Vt Vo) ie, about 55% of Vt. Blue dextran is also used for checking the column packing. A symmetrical peak of elution indicates homogeneity of packing. The inner volume (Vi) of the column can be determined by subtracting the void volume from the elution volume of small molecules such as glucose or tyrosine having K d -- 1.0. The total volume (Vt) of the column is the sum of void volume and inner volume. However, the total volume accessible to liquid is slightly less than the total volume due to a finite volume occupied by the gel matrix plus tightly bound water. Vt is equal to the volume of a cylinder whose height and radius are same as the dimensions of the column.

150

Sample size

2.0

This should not exceed 3% of the total volume and a smaller volume than this will give slightly better results down to about 1% of Vt. The starting protein concentration should ideally be in the range 10-20 mg/ml. A n increase in sample size will make the resolution poor. However, for desalting application of SEC 1 where the matrix has been chosen such that the desired protein elutes in the void volume while the elution volume of the salt approaches the total volume of the column, it is possible to use sample size as large as 20% of Vt.

~1.5

Sample application The operating buffer above the column bed is drained off and the clamp on the outlet tubing is closed. Then sample is applied gently to the column with a Pasteur pipette, allowing it to run down the side of the column while moving the pipette around the column. The stop-cock is then opened slowly and the sample allowed to pass down the upper surface of the gel. When all the protein sample has passed into the gel, buffer is applied in the same way and elution is performed with a constant flow rate. The column dimensions are important for resolution. Squat columns have fast flow rates and less turbulence leading to less spreading, but resolution is poor because the length covered will be smaller. In addition, it is difficult to achieve homogeneous sample application and buffer flow. On the other hand, a long thin column (20-40 times longer than its diameter) will give better results since the sample occupies a greater column depth. Fractions of appropriate size are collected and the column is monitored for protein using different methods (eg spectrophotometric, a dye binding method 5 or the method of Lowry et al. 6) Carbohydrate may be monitored using the method of Dubois et al. 7 Enzymatic activity may also be monitored. Eluting buffers should be of high enough ionic strength (eg >20 mM) to counteract the few charges which may be present on the gel. Apart from that, the only criterion for the buffer is that the proteins are stable in it.

1.0 I

s.o

s.4

LogM~ Figure 1 Plot of V e / V o v e r s u s log Mr for various marker proteins obtained on Sephadex G-150 column (52 x 1.7 cm) equilibrated with 0.06 M sodium phosphate buffer, p H 7.0. About 1.2 ml of the sample containing 10 mg of each of these marker proteins was applied. Elution was performed at a flow rate of 20 ml/h and fractions of 3 ml were collected. The column was monitored for protein using the method of Lowry et al. 6 The different marker proteins were: (1) a-chymotrypsinogen, (2) ovalbumin, (3) BSA monomer, (4) BSA dimer and (5) ~,-globulin 60

40

Applications of SEC

2O

Determination of Mr

SEC does not require pure, homogeneous protein. All that is needed is a method for detecting the protein as it comes off the column. For the determination of the Mr of any protein, a column is calibrated by passing proteins of known M r through the column. The elution volume of each protein is determined. These values of elution volume (Vc) are divided by void volume (Vo) to obtain (Ve/Vo), comparable on using different columns, for a particular protein. From the data obtained, a graph may be drawn between Ve/Vo and log Mr (Figure 1). The protein of unknown molecular weight is then passed through the column and its Ve/Vo used to estimate its Mr from the calibration curve. 8 SEC data can also be treated according to Porath. 9 Values for the elution volumes of marker proteins are normalised in terms of Kd values. A graph of Kd 1/3 and Mr v3 may be obtained using the values of marker proteins (Figure 2). Then the Kd value of t h e unknown protein is determined and hence its M r may be estimated. One source of error in this procedure is that it takes no account of molecular shape. In fact a protein molecule which is somewhat elongated will pass down the column faster than a spherical one of same molecular weight. Also it has been known for some time that glycoproteins, particularly those rich in carbohydrate, show anomalous behaviour on SEC and this will also be the case with proteins which are not globular. Nor is the technique applicable to proteins which have affinity towards carbohydrate. Nevertheless, the Mr derived by this technique with globular proteins seems to be accurate to about +10%. It is also possible to measure molecular weights under denaturing conditions provided appropriate standard curves are constructed. BIOCHEMICAL

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4"

014

0.6 '

o'.8

1'.o

Figure 2 Treatment of gel filtration data (as shown in Fig 1) according to Porath 9 Determination of hydrodynamic parameters

For the determination of the Stokes radius, a column is calibrated with marker proteins of known Stokes radii. The elution volumes of marker proteins are normalised into Kd and Kay values and these data are analysed according to Laurent and Killander 1° and Ackers 11 using the following equations: (-log

Kay) 1/2 :

A a + B

and a = ao + bo erfc -1 Kd where a is the Stokes radius of the protein with distribution coefficient Kd. A, B, ao and bo are calibration constants and erfc -1 Kd is the inverse error function complement of Kd. ~2 Both the above treatments yield straight lines (Figs 3 and 4). By substituting the values of ( - l o g Kay) v2 and erfc -1 Ks of the unknown protein, its Stokes radius can be determined. Values of the diffusion coefficient, D, and frictional ratio fifo of a protein may be calculated from the value for Stokes radius by using the following relationship 8 D = k T/(61r-qa)

fifo = a/(392 Mr/4"rrN) I/3

151 1.2

=>

3

v

4

co 0.8 O v

I

0A

Stokes radius (nm)

Figure 3 Treatment of gel filtration data (Fig 1) according to Laurent and Killanderw

/

A

E c

4

.E "lO

o.9 (n

2

1

0.4

I

1.0 e r f c -~ Kd

Figure 4 Treatment of gel filtration data (Fig 1) according to the method of Ackers 11 where k is the Boltzmann constant (1.386 x 10 -16 ergs/degree), T is the absolute temperature, -q is the coefficient of viscosity of the medium (0.01 poise for water and dilute aqueous solutions at 20°C), a is the Stokes radius of the protein, Mr is the molecular weight of the protein, 92 is the partial specific volume and N is Avogadro's number. SE-HPLC Although size exclusion chromatography has several advantages,

its major drawback is that it is time consuming. Separation times are long resulting in poor resolution. This is because of eddy diffusion, mass transfer problems and extra column effects. Speeding up the solvent flow by applying high pressure results further decrease in column efficiency and resolution. Application of high pressure causes the compaction of the bed due to the soft nature of the gel and results in lower flow rates. High pressure or high performance liquid chromatography (HPLC) involving the principle of size exclusion chromatography also known as size exclusion HPLC or S E - H P L C is an alternative approach for getting good resolution of macromolecules within a short time. Though primarily used for analytical purposes, the technique has now become a powerful purification technique. The fundamental principle remains the same for both SEC and SE-HPLC. The advantages offered by HPLC are good resolution and speed of analysis, reusability of column without repacking and regeneration, high reproducibility due to the close control of parameters effecting the efficiency of the separation, easy automation of instrument operation and data analysis and its adaptability for large-scale preparative procedures. These advantages of HPLC are the result of two major advances: (i) the development of new column packing material which is packed in narrow columns and which increases column efficiency 10-100-fold, and (ii) the improvement of elution rates achieved by applying high pressure (up to 300 atm). In general, S E - H P L C employs an immobile phase bonded onto a porous silica which allows high flow rates to be used. Semirigid gels can be used for fractionation of molecules up to a molecular weight of 10 000 000 under aqueous conditions. Rigid silica beads have several advantages over semirigid gels including ease of packing and compatibility with water and organic solvents. The new packings are typically spherical beads consisting of a solid non-porous core (40 I~m in diameter) with a thin, porous outer shell of absorbent (silica gel, alumina resin). Recently microparticulates (porous particles with diameters in the range 20-40 I~m and 5-10 Ixm) have been widely used because they offer greater resolution and faster separation with lower pressures. A variety of bonded phases have been used to mask the cationic surface of silica and prevent nonpermeation effects. These include glycerylpropyl diol and Nacetylaminopropyl silane. Although a number of nonsilica based support materials have been used, most work has involved the use of silica based material. Silica has the disadvantage of being unstable at pH values above 8.0. This can be overcome either by using a polymer coating or by surface stabilisation with zirconium which results in the development of rigid, cross-linked polymeric supports such as monobeads (Pharmacia) or TSK-PW (Toyo-Soda Company). Some of the column materials and their properties are listed in Table 1. For a successful HPLC separation, selection of both column

Table 1 S E - H P L C column materials available for protein purification* Column

Particle size (~m)

Pore size (nm)

Fractionation range (Ka)

pH stability

Superose - 12 Superose - 6 TSK 2000 SW TSK 3000 SW TSK 4000 SW Zorbax GF-250 Zorbax GF-450 Polyol=Si 300 Polyol=Si 500 SynChropak GPC 100 SynChropak GPC 300 SynChropak GPC 500

10 13 10 10 13 4 6 5,10 10 5 5 7

25 40 13 24 45 15 30 30 50 10 30 50

1-300 5-5000 1-50 5-400 40-1000 10-250 25-800 10-500 40-900 5-200 10-670 10->670

1-14 1-14 2.5-7.5 2.5-7.5 2.5-7.5 3-8.5 3-8.5 2-8.5 2-8.5 2-8 2-8 2-8

*Taken from ref 13 BIOCHEMICAL

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152 packing as well as solvent system is required. The mobile phase should consist of a very high purity solvent which should not react with sample or column packing and there should not be any interference with the detector. It has long been known that the polarity of a solvent dictates its ability to displace adsorbed solutes from the column ie eluting power, E, which increases with an increase in polarity. Sometimes the use of a single solvent does not give better resolution of solutes. This difficulty known as general elution problem is due to the wide range of Ka values of different components present in a multicomponent system. In such cases, solvent binary mixtures provide an extra advantage in resolving complex mixtures. Alternatively, the composition of the mobile phase can be varied starting with a weakly eluting solvent (low E) and gradually increasing the concentration of strongly eluting solvent (high E) in order to have gradient elution. The solvent are chosen for such type of gradient on empirical basis. In S E - H P L C where elution is carried out by using physiological buffers, the speed of S E HPLC can result in the quantitative purification of enzymes with full activity. However, for labile enzymes, S E - H P L C can be performed in the cold and at the highest possible flow rate. For those proteins which show aggregation, detergents generally 0.1% SDS can be included in the mobile phase.

References Wallach, J M (1989) In 'Practical Biochemistry for Colleges' (edited by Wood, E J) pp 63-64, Pergamon Press, Oxford eDixon, H B F (1985) Biochem Educ 13, 181-183 3Versee, V (1985) Biochem Educ 13, 33-34 4Malhotra, O P and Kumar, A (1989) Biochem Educ 17, 148-150 5Bradford, M M (1976) Anal Chern 72, 248-254 6Lowry, O H, Rosebrough, N J, Farr, A L and Randall, R J (1951) J Biol Chem 193, 265-275 7Dubois, M, Gilles, K A, Hamilton, J K, Rebers, P A and Smith, F (1956) Anal Chem 28, 350-356 8Andrews, P (1970) Methods Biochem Anal 18, 1-53 9porath, J (1963) J Pure Appl Chem 6,233-234 mLaurent, T C and Killander, J (1964) J Chromatog 14, 317-330 1~Ackers, G K (1967) J Biol Chem 242, 3237-3238 t2In 'Tables of the error function and its derivative', NBS Applied Mathematics Series 41, United States Government Printing Office, Washington, DC, 1954 X3Welling, G W and Welling-Wester, S (1989) In 'HPLC of Macromolecules - - A Practical Approach', pp 77-89, IRL Press/OUP, Oxford

procedure involves fluorescence using fluorescein mercuric acetate 3 and a reduction with sodium borohydride. 4 The fluorescence procedure was chosen to complement the very strong instrumental bias which students get in their applied chemistry practical course. Students are told about the great sensitivity of fluorescence, the requirements of separate excitation and emission wavelengths and the phenomenon of quenching which are involved in fluorescent assays. The experiment is run in the final year of the course because of the potential toxicity of fluorescein mercuric acetate 3 and the care required with the powerful reducing agent sodium borohydride.5 As described, the work may be performed in less than four hours by a pair of students.

Materials and Methods Solutions All protein solutions are made up in distilled water at a concentration of 1 mg/ml. Fluorescein mercuric acetate is maintained at 10 -4 M in 0.01 M NaOH but before use is diluted to 10 -5 M with 1 M NaOH. Ellman's reagent, 5,5'-dithiobis-(2nitrobenzoic acid), is made at 10 mM in 0.05 M phosphate buffer pH 8, and a 1 M solution of KH2PO 4 containing 0.2 M HC1 is also prepared and kept as a stock solution. Fluorescence Assay A solution of crystalline RNase is used as a standard and the solution containing 1 mg/ml is diluted 1 + 30.7 with distilled water to contain 10 nmol of disulphide groups per ml (2.5 nmol or 31.5 ~g of protein). Standard 1 ml solutions are then prepared from this dilution to contain 1-10 nmol of disulphide groups. Other protein solutions are diluted to contain approximately 5 nmol of disulphide per ml. The dilutions used are: 1 + 57 for lysozyme (M r 13 930), 1 + 49 for trypsin (Mr 23 800), and 1 + 54 for bovine serum albumin (Mr 65 900). One ml volumes of the final dilution are used. One ml of 10-SM fluorescein mercuric acetate solution is added to each tube, followed by 8 ml of 1 M NaOH. Tubes are vortexed and left for 1 h prior to determination of fluorescence intensity in each tube. Fluorescein mercuric acetate shows intense fluorescence. Thiol groups react with this reagent under both alkaline conditions and at neutral pH whereas disulphides only react at alkaline pH. A correction is required when thiol groups are present in the protein. The reaction is presumed to involve a relatively slow alkaline scission of disulphide bonds and a very rapid formation of a complex between the thiol groups formed and fluorescein mercuric acetate to quench the colour. There are probably irreversible side reactions as well.

Disulphide Groups in Proteins (CH~COO)Hg~ v

W L BAKER and A PANOW Chemistry Department Swinburne Institute o f Technology John Street Hawthorn 3122, Melbourne Australia

Introduction Disulphide groups in proteins may have a role in metabolic activity and control ~ or in the interaction of hormones with their receptors. 2 However, teaching at the undergraduate level tends to concentrate primarily on their stabilising role on the tertiary structure of proteins and peptides. In either case it is necessary to have adequate analytical methods to determine the number of disulphide groups in proteins. The final year biochemical practical unit in this Institute contains an experiment which compares two methods of analysis of disulphide groups. The BIOCHEMICAL

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~

-CJ~'~.~'~,.Hg(OOCCH0 COOH

Students are required to determine the wavelengths of maximum emission and excitation, which are about 525 and 480 nm respectively, and draw the appropriate curves. Although procedures vary with the instrument the principle is general for all instruments. The emission wavelength is determined using a fixed excitation wavelength (usually 300 nm) and variation in sensitivity or attenuation of the instrument. The excitation wavelength is determined by scanning from the wavelength of maximum emission. Results are obtained from the standard curve of RNase and the number of moles of disulphide per mol of protein are determined by working back through the dilutions.

Reducing assay

Lysozyme is diluted to a concentration of 0.5