Rapid analytical gel filtration chromatography

ANALYTICAL

BIOCHEMISTRY

Rapid

61, 161-111 (1974)

Analytical

III. Apparent

Gel

Chromatography

Molecular Weight Distribution Produced by Proteolysis

NICHOLAS Laboratory Massachusetts

Filtration

of Peptides

CATSIMPOOLAS

of Biophysics, Department Institute of Technology,

of Nutrition

Cambridge,

and Food Massachusetts

Science, 02139

Received December 20, 1973; accepted March 8, 1974 A rapid gel filtration chromatography method is described for determination of the molecular weight distribution (MWD) of peptide mixtures by using calibrated Sephadex microbore columns. The method was applied to MWD analysis of peptide mixtures resulting from trypsin and pepsin digestion of glycinin-the major soybean storage protein-under different incubation conditions of pH, temperature, and time of hydrolysis. Possible sources of errors and suggestions for improvement are discussed.

Gel permeation chromatography (GPC) which employs semirigid cross-linked polystyrene beads (1) has been used extensively for the determination of the molecular weight distribution (MWD) of synthetic organic polymers (2). However, gel filtration chromatography (GFC) which uses soft cross-linked dextran beads (3) has only recently been applied to similar determinations involving MWD of synthetic collagen polypeptides (4) in regular size columns. With the recent development of rapid analytical gel chromatographic techniques (56) utilizing microbore columns packed with Sephadex G-56 and G-25 gels, MWD determinations in aqueous system may be of importance in certain areas of research. In this laboratory, a need was realized for the estimation of the relative size of peptide fragments and aggregates resulting from the enzymatic digestion of food proteins of plant origin. This information has important implications in nutritional studies and the development of proteins with desirable functional characteristics in human food. Susceptibility of proteins to attack by proteolytic enzymes is usually measured by determination of peptide bonds cleaved either colorimetritally or with the pH-stat procedure. Other methods such as ultracentrifugation, dilatometry, viscosimetry, optical rotation, and electrophoresis have been used less frequently. Since proteolytic cleavage depends on both 101 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

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CATSIMPOOLAS

enzyme specificity and availability of peptide bonds, information about the MWD of the peptides produced should be valuable in probing the structure of native and denatured proteins. In the present study, we have investigated the use of a rapid analytical gel chromatography technique (5,6) for the determination of the apparent MWD of peptides produced by the action of trypsin and pepsin on glycinin, the major soybean storage protein, under different conditions of temperature and pH. Apparent molecular weight averages have been obtained by adoption of gel permeation chromatography expressions (2) with appropriate standardizations and modifications for the present system. An important advantage of the present method is that an analysis can be completed in 40 min with as little as 5 pg of protein material (6). EXPERIMENTAL

Muter-ids. Glycinin (the major “storage” soybean protein) was prepared in homogeneous form by combination of ammonium sulfate fractionation and DEAE-Sephadex chromatography as described previously (7). Trypsin (2X crystallized) was obtained from Worthington. Chymotrypsinogen A, cytochrome c, and ribonuclease were obtained from Calbiochem. Pepsin, bacitracin, insulin A, thyroglobulin, and dinitrophenyl alanine were products of Schwarz/Mann. Polymixin B, insulin, and achymotrypsin were obtained from Sigma. Sephadex G-50 (medium) was purchased from Pharmacia. Enzymatic hydrolysis. Proteolysis of glycinin with trypsin and pepsin under different experimental conditions of pH and temperature were carried out in a London Radiometer pH-stat system. The system included: (1) type ABU 12 Autoburette; (2) type SBR 2 titrigraph; (3) type TTA 31 titration assembly with thermostatting jacket-vessel; (4) type TTT 11 titrator module; (5) type PHM-26 expanded scale pH meter; and (6) model T6K water circulator. In this particular experiment the concentration of glycinin was 2.0 mg/ml. At the termination of the enzymatic digestion experiment, the hydrolysate was freeze-dried and redissolved in pH 7.6 (0.1 M in NaCl) phosphate buffer (7) for chromatographic analysis. Gel chromatography. The analytical gel chromatography experiments were performed as described previously (5,6). The main components involve a Chromatronix microbore column (MB-3-500)) sample injection tee (107B25), and Cheminert fittings. The flow rate was regulated with a Milton Roy instrument minipump. The effluents were monitored (at 220 nm) with a Gilford 2000 recording spectrophotometer, a Beckman monochromator, and Gilford microflow cells. As described previously for transient isoelectric focusing experiments (8)) the entire concentration pro-

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CHROX~T~GRAPHT

103

file can be digitized at regular time intervals with an Infotronics model CRS-208 automatic digital integrator (with digitizing accessory). Statistical moment analysis of the absorbance distribution of a peak is performed as described in a recent report (9). In the absence of automatic digitizing equipment, the absorbance values (Ai) at the ith peak interval are read directly from the calibrated recorder chart at constant time intervals (ti). Baseline corrections and statistical peak analysis are again performed as described (9). The manual procedure for obtaining the numerical value of the data points is tedious and subject to errors of personal judgment. The relationship between number of data points collected per peak, percent digitizing limit, and errors in the calculation of moments of peaks of different shape have been discussed for a general case (9,lO). PRELIMINARY

CONSIDERATIONS

In order to measure the MWD of components in a given sample by gel chromatography (either by GPC or by GFC) the following minimum requirements should be ideally met: 1. The solute is soluble in the mobile phase (i.e., elution buffer or solvent). 2. There is no aggregation or dissociation of the solute during elution. 3. The absorbance [or other property, e.g., optical rotation (4) ] of each component in the mixture is directly proportional to the amount of the component present. 4. The solute distributes itself between the upper and lower limits of retention (to and tn) in a given gel. 5. The substances employed in the calibration of the column are as closely related as possible (in relation to composition and shape) to the material under analysis. 6. An empirical mathematical relationship exists between molecular weight (M) and retention time (te) for a particular component in a given gel (column calibration). 7. There is no physical/chemical interaction of the solute either with the mobile or stationary (gel) phase other than the exclusion process implies. Thus, there is no adsorption, ionic binding, or covalent bond formation. 8. All species of a given molecular weight are eluted in an infinitely sharp band of Gaussian shape. 9. The sample represents a multicomponent mixture. 10. The standard deviation (a) of the concentration distribution of each component in the mixture is the same for all components.

104

NICHOLAS

THEORETICAL

CATSIMPOOLAS

BACKGROUND

Methods for the determination of molecular weight averages have been discussed in general references (11-13) which should be consulted for detailed theoretical background information. The number-average moIecular weight (*,J can be determined by osmotic pressure measurements, the weight-average molecular weight (@,) by light-scattering experiments, and the z-average molecular weight (J?,) from equilibrium sedimentation data. When colligative properties are utilized, each molecule, large or small, makes the same contribution to the observed averaging process. If the total weight were shared equally among the molecules in the mixture, the observed contribution will remain unchanged thus leading to the determination of a,. However, the large species are weighted more heavily in the weight average than in the number average, so that for a poIydisperse sample aw > .L@~.The fraction i@,/Bn is used to characterize the “polydispersity” of the sample. Very high molecular weight species influence more the z-average than the weight average. Gel filtration chromatography can be used to determine simultaneously A%,, a,, and L@,, which is a very attractive feature of the method considering the simplicity and rapidity of the experimental procedure. However, GFC being a secondary method requires calibration against a primary method (e.g., osmometry, light scattering, sedimentation) of molecular weight determination, and therefore the use of molecular weight markers. The molecular weight distribution is characterized by its principal statistical moments and is given by: iit, = 1 niMi/ i=l

2 ni i=l

(3)

where ni is the concentration number of molecules of the ith kind per unit volume and Mi is their molecular weight. In addition, the partition coefficient (iL,) in rapid gel filtration experiments is given by (5) : Kav = (te.- to)/(t, - to),

(4)

where to is the minimum retention time for the column determined with a totally excluded compound (e.g., thyroglobulin), t, is the maximum retention time measured with a totaIly included compound (e.g., dinitro-

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105

CHROMATOGRAPHY

phenyl alanine) ~and t, is the retention time of any substance which satisfies the condition t,, < t, < t,. The marker proteins and peptides used in this study obey the empirical relationship (5) : -log K,, = RM2’3 + S,

(5) where M is the molecular weight of the marker, and R and S are constants obtained by linear regression analysis. In this work R = 9.78 X 1R4 and S = 2.14 X 1e2 for a Sephadex G-50 gel. Combination of Eqs. (4 and 5) produces :

Considering ti to be the ith time interval-of the molecular weight distribution-at which Mi (the MW at the ith interval) is measured, then Eq. (6) becomes:

Assuming the standard deviation (u) of the of all species to be identical, the concentration ecules in the ith interval is proportional to the (6). Therefore, the proportion of the total fraction is A i@t) lli = ZAi(at)’

concentration distribution of protein or peptide molabsorbance (Ai) at 220 nm weight present in the ith

where At is the constant interval at which Ai measurements So that Eqs (1, 2, and 3) become: ZAi Mn = ZA;/‘M;

were taken.

(9) (10) (11)

Combination of Eqs. (7, 9, 10, and 11) produces the final equations for calculation of L@‘,, a,, and %, as follows: (12)

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NICHOLAS

CATSIMPOOLAS

(13)

(14)

In practice, Ai at ti can be obtained automatically by digitizing the absorbance at a constant time interval (At). S and R are predetermined calibration constants obtained by linear regression from Eq. (5). The values of t, and t, are obtained from the first statistical moment of the unretained and completely retained peaks, respectively, so that: t = 0

[ 1 ZAiti - ZAi

peak 0

05)

This process assumes that the two marker peaks (distributions) are monodispersed (i.e., i@,/i@, = 1). Equations (12-14) can assume other forms depending on the relationship between the partition coefficient and molecular weight of marker compounds, and whether this is assumedto be linear or polynomial (14). Third degree polynomial fitting techniques have been shown recently (14) to offer a much greater degree of precision (i.e., as low as 0.1%) than linear equations in the analysis of retention data of globular proteins subjected to gel filtration. If a polynomial fit is adopted, MWD analysis will require the use of a digital computer for on-line data processing. RESULTS

AND

DISCUSSIONS

A typical elution profile of glycinin peptides obtained by tryptic hydrolysis at pH 8.0, 37”C, Z-hr hydrolysis is shown in Fig. 1A. Five micrograms of the mixture was used to avoid overloading of the Sephadex G-50 microbore column. The analysis is complete at 40 min, an attractive feature of the present technique. The concentration distribution of the peptide mixture expressed in terms of molecular weight (M) rather than retention time (te) is illustrated in Fig. 1B. The points in the distribution corresponding to the apparent &l,, aw, and i@, values obtained by using Eqs. (12-14) have been marked. The sametype of analysis

RAPlD

FIG. 1. Gel filtration from a 2-hr, pH 8.0, absorbance: 220 nm; 7.6 phosphate (0.1 M Differential molecular

GEL

107

CHROMATOGRAPHY

chromatographic analysis of a glycinin hydrolysate resulting 37”C, trypsin digestion experiment. Column: Sephadex G-50; sample: Five micrograms of the peptide mixture; buffer: pH in NaCl) ; flow rate 0.083 ml/min: (A) Elution profile; (B) weight distribution.

was performed t,o derive the apparent values of i@,,@,,i@,, and A?,/i@, of glycinin peptide mixkes incubated with trypsin either at constant temperature (37°C) and different pH (Table 1) or at pH 10 and variable temperature of incubation (Table 2). The results of a time course experiment using pepsin at pH 2.0, 37°C to hydrolyze glycinin are depicted in Table 3. General observat,ions from these experiments can be summarized as follows. Pepsin hydrolysis produced pept.ide mixtures of considerably TABLE 1 Apparent Molecular Weight Distribution of Glycinin Peptides Obtained by a 2-hr Tryptic Hydrolysis at 37°C and Different pH Values M x lo+

8.0 8.5

8.9

9.0

10.3 8.9

9.5 10.0 10.5 11.0

10.1 11.0 11.8

14.3 13.2 13.3

1.13 1.26 1.14

11.3 9.6

13.0

1.26

7.4

11.8

1.29

7.6 8.8

9.9 11.0

12.2

1.30

13.2

1.25

8.7

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NICHOLAS

CATSIMPOOLAS

TABLE 2 Apparent Molecular Weight Distribution of Glycinin Peptides Obtained by a 2-hr Tryptic Hydrolysis at pH 10 and Different Incubation Temperatures Temp “C 22 27 32 37 42 47

9.7 9.7 10.0 7.4 9.0 11.0

11.9 11.8 12.2 9.6 11.5 13.2

14.1 13.9 14.3 11.7 13.9 15.3

1.22 1.21 1.22 1.29 1.27 1.20

apparent il?l,, i@,, and n;i, values, and higher i@,,,/i@, polydispersity factor than trypsin hydrolysis of the same duration and temperature. The greater extent of pepsin hydrolysis occurred within 1-hr incubation period, since the apparent MWD values did not change appreciably at longer times. The effects of pH and temperature of incubation on the 2-hr tryptic hydrolysis of glycinin were not dramatic. However, optimum conditions (i.e., corresponding to the lowest icn, fi,, and &yZ values) were obtained at pH 10 and 37°C which agrees with independent pa-stat reaction rate studies (unpublished data). Assuming that lysine and arginine residues are uniformly distributed, and that the polypeptide chains of glycinin are readily available to proteolytic attack, the size of the tryptic peptides should have been in the range of 1000 daltons. Glycinin has a molecular weight of approximately 350,000 and a total 136 lysine and 178 arginine residues (15). The experimentally observed MWD values are approximately ten times higher than the expected. This indicates that under the present experimental conditions only approximately one-tenth of the potentially reactive peptide bond sites of glycinin are split by trypsin. These findings are in general agreement with spectrophotometric titration

lower

Apparent

Time (hr) 1 2 4 16

TABLE 3 Molecular Weight Distribution of Glycinin Peptides Obtained by Pepsin Hydrolysis at pH 2.0 and 37°C 5.0 5.3 4.3 4.2

7.0 7.2 6.2 5.9

9.1 9.4 8.0 7.8

1.40 1.35 1.44 1.40

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109

data (15) which show that. only 10% of the tyrosine phenoxy groups of glycinin can be titrated even up to pH 11.0. The protein can be unfolded significantly only above pH 11.0 or in the presence of dissociating agents such as 6 M urea or 4 M guanidine hydrochloride (16). It should be noted that in the present experiments the amount of the enzyme was fixed and no attempt was made to examine the possible relative loss in activity at high pH values. It is also possible that the peptides may associate to higher molecular weight species, although not likely because in such a case much higher MWD may have been observed. MWD experiments on enzymatic protein hydrolysates under controlled conditions of pH, temperature, time, and type and concentration of enzyme may offer valuable information in regard to the (a) relative size of peptides, (b) structural susceptibility of the protein to proteolysis, (c) recognition of readily hydrolyzed part of a molecule in relation to a remaining high molecular weight inert core, and (dl polydispersity of the peptide mixture. One of the vulnerable assumptions in the present system is that the compounds used for column calibration (small globular proteins and polypeptides) are all of the same shape and amino acid composition among themselves, and with the polypeptide fragments under investigation. The magnitude of error in the determination of the absolute MWD under this assumption is unknown. However, the calibration curve can be considered as a ‘(reference” for comparison of “apparent” MWD of peptides derived under different conditions of proteolysis (e.g., pH, temperature). Assuming that the resulting polypeptides vary little in shape and composition among themselves, the “apparent” average molecular weight values offer information at least in regard to the relative extent of proteolysis. This approach offers a first approximation in the implementation of the method as an analytical tool. Otherwise the search for absolute calibrants ideally suited to each particular protein digest will render the technique impractical. An alternate solution to the column calibration problem is the use of dissociating media (17-20) so that, a]] components assume “random” conformations. This approach also assumes similar amino acid composition. However, in the present particular case the use of dissociating solvents or detergents was not considered desirable, since the contribution of possible aggregation to the apparent M-T) of the proteolytic products is of value in assessing their nutritional and/or functional performance. In future studies, comparison between the two methods will be of interest. An additional vulnerable assumption is that peptide species of a given molecular weight are eluted as infinitely sharp bands of Gaussian shape having identical standard deviation (a). In practice, the bands are broad,

110

NICHOLAS

CATSIMPOOLAS

probably not of perfect Gaussian shape and u is increasing (band spreading) as a function of the retention time. The spreading of the zones occurs because of (a) flow velocity unequalities (tortuous path) depending upon packing structure and the relative position of the stationary phase particles, (b) molecular diffusion in the longitudinal direction, (c) nonequilibrium of solute between the mobile and stationary phases, and (d) instrumental factors. Corrections for these phenomena will be extremely complex and virtually impossible at the present state of the technique. It is also interesting to note that if a single species is analyzed by the present method and the data are plotted as shown in Fig. lB, it will appear as being composed of several species of different molecular weight; an erroneous conclusion. Assuming that if the peaks of different species-under a cumulative Gaussian distribution-are separated by less than 1 SD their distributions will be overlapping, the minimum number of bands required to perform MWD analysis should be four to five for an apparent Gaussian distribution. Reduction in packing-particle size and use of small column diameters, with porous glass packings have been recently shown to increase the efficiency of gel permeation chromatography (21). Similar developments in gel filtration chromatography combined with increased speed of analysis, computer processing of data, and study of the factors affecting zone spreading and resolution will be very desirable. Although many improvements are needed in order to realize the full potential of the technique, the experiments reported here and in a recent report (4) are the first attempts to use gel filtration for MWD determinations and to point out some of the assumptions, advantages, and difficulties. Reduction in packing-particle size and development of new porous materials may increase the efficiency of filtration chromatography and reduce the time of analysis to a few minutes. This approach has yielded significant increases in gel permeation efficiency (21). Similar developments in this field combined with careful selection of standard calibrants and computer processing of data will provide good accuracy and precision in MWD analysis of biological materials. Furthermore, the adoption of precise scanning gel chromatography techniques (22) will offer an additional advantage for analytical gel filtration systems over other types of column chromatography where the opaque nature of the packings prohibits the use of in situ uv absorption methods. REFERENCES 1. MOORE, J. C. (1964) .I. Polvm. Sci. Part A 2, 835. 2. OTOCKA, E. P. (1973) Act. Chem. Res. 6, 348. 3. PORATH, J., AND FLODIN, P. (1959) Nature (London)

183, 165’7.

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4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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FAIRWEATHER, R., JONES, J. H., AND WILCOX, J. K. (1972) J. Chromatogr. 87, 157. CATSIMPOOWS, N., AND KENNEY, J. (1972) J. Chromatogr. 64, 77. CATSIMPOOLAS, N., AND KENNEY, J. (1972) J. Chromatogr. 71, 573. CATSIMPOOLAS, N., ROGERS, D. A., CIRCLE, S. J., AND MEYER, E. W. (1967) Cerenl Chem. 44, 631. CATSIMPWLAS N. (1973) And Biochem. 54, 66. C.~TSIMPOOLAS, N., AND GRIFFITH, A. L. (1973) Anal. Biochem. 56, 100. CHESLER, S. N.. AND CRAM, S. P. (1971) Anal. Chem. 43, 1922. TANFORD, C. (1961) Physical Chemistry of Macromolecules, Chapters 4-6, WileyInterscience. New York. BILLMEYER, F. W. (1962) Textbook of Polymer Chemistry, Chapter 1, Interscience, New York. MORAWETZ, H. (1965) Macromolecules in Solution, Chapter 1. Interscience, New York. GROVER,A. K., AND KAPOOR, M. (1973) And. Biochem. 51, 163. CATSIMPOOLAS, N., BERG, T., AND MEYER, E. W. (1971) Znt. J. Protein Res. 3, 63. CATSIMPOOLAS, N., WANG J., AND BERG, T. (1970) Int. J. Protein Res. 2, 277. PUSZTAI, A., AND WATT, W. B. (1970) Biochim. Biophys. Acta 214, 465. DAVISIN, P. F. (1968) Science 161, 906. FISH, W. W., MANN, K. G., AND TANFORD, C. (1969) J. Biol. C&m. 244, 49%. FISH, W. W., REYNOLDS, J., AND TANFORD, C. (1970) J. &o/. Chem. 245, 5166.

21. OTOCKA,

E. P. (1973)

J. Chromatogr.

76, 149.

22. BRUMBAUGH E. E., AND ACKERS, G. K. (1968) J. Biol. Chem. 243, 6315.