Metal affinity electrophoresis: An analytical tool

METHODS:

A Companion

to Methods

in Enzymology

4, 97-102

(1992)

Metal Affinity Electrophoresis: E. Nanak, J. Abdul-Nour, Laboratoire CompiZgne,

de Technokie BP 649, 60206

An Analytical Tool

and M. A. Vijayalakshmi’

des S&war&ions. D@wartement > CompiGgne, France

GEnie Biologique,

Immobilized metal ion affinity electrophoresis, a merger of the two concepts of affinity electrophoresis and immobilized metal ion affinity chromatography, is a useful analytical tool for the quantitative determination of binding constants between protein and chelated metal ion, for the efficient design of preparative-scale purification of proteins using chromatographic or phase partition techniques, for the detection of protein unfolding, and for the eventual study of structure/ function relationships of modified proteins. The optimal conditions and the “modus operandi” of the system are described. o ww Academic PESS, I~C.

Affinity electrophoresis (AE) merges the principle of electrophoretic separation and the interaction of a biomolecule with a ligand immobilized within the electrophoresis gel support. This concept is widely used with biospecific ligands, lectins, and triazine dyes (biomimetic) (l-3). The principle of immobilized metal ion affinity (IMA) has been only recently exploited in AE (4,5). Although the extension is logical, a few questions remain to be addressed about the proper design of experiments for increasing its utility as an analytical tool at the upstream or even the downstream of fine chromatographic steps such as immobilized metal ion affinity chromatography (IMAC).

‘To whom 44.20.48.13.

correspondence

should

1046.2023/92 55.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

be

addressed.

Fax:

(33)

Uniuersitk

de Technologie

de

BASIC PRINCIPLES It is well known that IMAC functions mainly through an electron donor-acceptor (EDA) mechanism whereby the metal ion immobilized through a chelating agent (in most cases iminodiacetic acid (IDA)) to an insoluble matrix acts as an electron acceptor and the imidazole nitrogens of the accessible histidines on the surface of a protein act as the electron donors (6). Several systematic investigations of IMA interactions both in IMAC and in immobilized metal ion affinity partition (IMAP) have revealed a direct relationship between the number of unprotonated accessible histidine residues and the strength of protein binding to the polymer-supported copper or zinc ions (7-9). Therefore, if metal affinity electrophoresis (IMAElec) is a direct extension of the generic IMA principle, this relationship should be maintained regardless of the applied electrical field. This will depend directly on the relative stability constants between the metal ion and the chelator used and the metal-chelate protein complex. Moreover, the mode of inclusion of the affinity ligand (chelated metal ion) into the electrophoretic gel can influence the stability of the ligand as well as its distribution in the gel. Furthermore, whether the metal chelate (IDA-Me(H)) as such, or the same coupled to a polyhydroxylic polymer, should be considered the interacting ligand has not been determined. This is very pertinent when we consider the influence of the microenvironment of the metal ion on its strength of binding to a given protein (5). In recognition of these different aspects, we discuss the technical approaches and the applications of IMAElec. 97

98

NANAK,

ABDUL-NOUR,

such as Sepharose with coupled ble polymers such as polyethylene IDA-Me(I1) (5).

HOW TO DO IMA-ELEC Preparation of Ligand-Incorporated Gels

AND VIJAYALAKSHMI

Electrophoretic

Preparation

Choice of Support for Electrophoresis The two most prevalent polymer supports, agarose and polyacrylamide, used in conventional electrophoresis can be considered as supports. However, agarose scores better than the latter because (i) its large pore size is compatible with large protein molecules, and eventually with DNA, for studying metal-DNA interactions; (ii) no complicated chelator coupling and crosslinking chemistry are required; and (iii) any eventual quenching of the EDA interactions involved in metal-protein recognition, as may be the case with polyacrylamide supports, is avoided. Incorporation of the Interacting Metal Ligand The interacting metal ligand is in fact the metal ion coordinated to a chelating group such as IDA, Triscarboxymethylated ethylenediamine (TED), or nitrilotriacetate (NTA). Two approaches for incorporating the chelated metal ion into the electrophoretic gel are possible: (i) chemical coupling of the IDA-Me(I1) to the electrophoretic gel itself, and (ii) incorporation of the polymer coupled with IDA-Me@) into the agarose gel before the electrophoretic gel casting. The latter approach seems more attractive both because of its simplicity and because of the drawbacks observed with the first approach during its use with other ligands (10). Incorporation of the polymer-supported IDA-Me(I1) into the agarose electrophoretic gel can be accomplished by using either insoluble particulate polymers

of pH

and

IMA-Elec

Supporting with

pH 8.2 RNase A B Cytochrome c Candida krusei Horse heart Tuna heart Note. ND, not determined; Kd values = 20%.

Polymers

Sepharose pH

With PEG 5000-IDA-Me(II) as Ligand PEG 5000-IDA-Me(I1) was synthesized as described in (8) using monomethoxy PEG 5000 as the starting material and a two-step chemical reaction consisting of the preparation of amino-PEG 5000 and derivatization into iminodiacetate PEG 5000 using bromoacetic acid. Alternatively, direct derivatization of Cl-PEG 5000 into IDA-PEG 5000 was also performed according to the method described in (9). The chelated PEG was then metallized as described in (8). This soluble polymer containing the affinity ligand, IDA-Me(H), in the concentration range of 0 to 2.5% (w/v) of PEG 5000IDA-Me(I1) representing metal concentrations in the range of 1.0 to 5.0 mM, was mixed into the 1 or 2% agarose gel and melted at 70°C.

Selection of Optimal Electrophoretic

6.25 mM 11.10 mM

4.07 mM 4.74 mM

8.20 mM Abnormal 20.70 mM

3.72 mM 10.96 mM

BT,

below

148.00mM threshold,

i.e., the value

Conditions

Selection of optimal electrophoretic conditions (current intensity, pH) and the relative migration of

1

on the

Affinity

of Model

Proteins

GB-IDA-Cu(I1)

7.2

Agarose Gels

With Sepharose GB-IDA-Me(U) as Ligand Insoluble chelating Sepharose GB-IDA-Me(I1) beads were added to a 1% (w/v) agarose solution in the corresponding buffer at a selected pH, by adding varying amounts of suction-dried, metallized, Chelating Sepharose in the range of 0.5 to 3.5 g of the gel representing the metal (e.g., Cu(I1)) concentration 3.0 to 20.0 mM). Then 3.5 to 10 ml (depending on the size of the electrophoresis plate) was poured and spread on a sheet of gel-bond film (8 X 4 cm or 12.3 X 5 cm) and used as described in (4).

TABLE Influence

of Electrophoretic

ligand or soluglycol carrying

IMA-Elec pH

5.5

ND ND

PEG pH

69.00 mM BT

67.00

zero. The

maximum

5000-IDA-&( 7.2

pH

22.00 mM

BT BT BT

mM mM

of (do - d) is near

with

pH 8.2

45.00 mM 66.70 250.00

to IDA-Cu(I1)

mM

BT BT

mM mM 400.00mM

BT BT BT

63.00 160.00

error:

5.5

migration

distances

= 4%.

METAL

AFFINITY

the free protein and the protein interacting with ligand are essential for reliable quantitative determination of the affinity and ultimately the eventual usefulness of IMA-Elec. Optimal Applied Current Intensity Results from our previous studies of the influence of applied current intensities in the range of 8 to 52 mA have shown that current intensities up to 20 mA were compatible with a linear relationship in the double inverse plot of the differential migration distance (do - d) and the ligand concentration (L). In contrast, when current intensities of 30 and 50 mA were used, deviation from the linearity of the l/d,, - d vs l/L graph and also increased migration of the protein having higher affinity for the ligand (IDA-Cu(I1)) (inactivated chymotrypsin) in the presence of the ligand, compared to the blank, were seen. This resulted in a negative linear graph of l/d0 - d vs l/L for this protein (5). This could be attributed to the fact that ligandprotein complex formation becomes the limiting factor, similar to what has been shown by Poulsen et al. (11) in the case of enzyme-substrate AE. Moreover, in IMAElec the stability of metal coordination can be jeopardized at high current intensity. This was confirmed by the presence of copper leakage out of the gel when a 50-mA current was applied. Hence, the optimal working current intensity for achieving a good linear graph of l/d, - d vs l/L for dissociation constant (&) determinations is 20 mA.

ELECTROPHORESIS

99

ever, at pH values above 8.2 the basic proteins (cytochrome c variants) showed a decrease in their affinities for IDA-Cu(I1) in IMA-Elec. In the case of acidic proteins such a decrease was not a general phenomenon. However, the range of pH values at which the systems can be run will depend on the relative migration of the free protein and the protein-ligand complex at the given pH. As is shown in Table 1, pH 7.2 seems to be optimal in general. Table 1 summarizes the influence of different parameters, such as the nature of the supporting polymer and the electrophoretic pH, on the KD values determined for a few model proteins previously studied by IMAC or IMAP. Electrophoretic Buffer Three different buffers were studied for use in IMAElec: Tris-acetate, sodium phosphate, and sodium acetate. At acidic pH, bovine ribonuclease can be studied only with acetate buffer because phosphate ions bind to the active site of the enzyme. Tris-acetate, 0.1 M, pH 7.2, was selected because the optimum operating pH in IMA-Elec is near neutral pH. Sample Application The amount of protein to be injected should be compatible with two basic rules: (i) The quantity of protein

Electrophoretic pH Preliminary studies carried out with several model proteins and with two types of polymer-supported IDACu(I1) have shown an increase in affinity (decreasing KD values) for these proteins from pH 5.5 to 7.2. How-

TABLE Typical

Migration

Distances

2 of Native

Bovine

Pancreatic

Ribonuclease A with and without the Affinity Ligand [ IDA-Cu( l/L

(mMml) 0.20 0.25 0.33 0.50 1.00

o An average

do (cm)” 2.25 2.35 2.35 2.50 2.60

of five

t + f IL f

0.05 0.05 0.05 0.05 0.05

experiments.

II)] d (cm)” 1.25 1.45 1.65 2.00 2.35

f f 5 2 +

0.05 0.05 0.05 0.05 0.05

l/(d,

- d) (cm-i) 1.00

1.10 1.42 2.00 4.00

FIG. 1. Affinity electrophoretic migration of proteins on ligand (IDA-Cu(II))-incorporated agarose gels: (A) Without ligand, in the presence of 5 mM PEG 5000. (A’) With ligand in the presence of 5 mM PEG 5000-IDA-Cu(I1). Lane 1, native ribonuclease A, 2, ribonuclease A denaturated by 6 M guanidinium chloride; 3, ribonuclease A denaturated by 8 M urea. (B) Affinity electrophoresis of a mixture of native and denatured ribonuclease A: Lane 1, in the presence of 2 mM PEG 5000; 2, in the presence of 2 mM PEG 5000-IDA-Cu(I1); 3, in the presence of 4 mM PEG 5000; 4, in the presence of 4 mM PEGi000-IDA-c~(11).

100

NANAK,

ABDUL-NOUR,

should not saturate all interaction sites and should permit the formation and dissociation of the ligandprotein complex. (ii) The protein content of a sample should be sufficient to be detected by Coomassie blue staining but should also be as small as possible to form a fine band in agarose gels. With Coomassie blue staining, 5 pg of protein seems to be appropriate. Typically, samples containing 5 yg of protein in 4-20 ~1 volume are introduced into the wells. The ionic strength of the samples should be approximately the same as that of the electrophoresis buffer. To ensure a reasonably uniform diffusion time for all samples in an experiment, the time between application of the first and the last sample should not exceed 10 min. General Rules for Choosing Electrophoretic

Conditions

Buffer: Tris-acetate, 0.1 M, pH 7.2 Electric field: 5 V/cm-Samples (5 pugin 10 ~1) Time: 2-7 h depending on the size of the electrophoresis plates. Staining: 10 min with 0.5% (w/v) Coomassie brilliant blue G-250 in methanol:acetic acidwater (4:1:5, v/v) Destaining: 3 h with methanol: acetic acidwater (4:1:5, v/v) Table 2 gives a brief summary of typical migration distances measured on vertical electrophoretic gels af-

AND

VIJAYALAKSHMI

ter Coomassie blue staining. The migration distances represent experimental values obtained for native bovine pancreatic ribonuclease A as used in Fig. 2 for the calculation of its dissociation constant.

WHAT BASIC INFORMATION CAN IMA-ELEC GIVE? 1. A quantitative expression of the strength of binding using a Takeo-Bog-Hansen plot (2) with very small amounts of protein is feasible. Examples are shown in Table 1. This method thus has an advantage over the frontal elution techniques used in IMAC. 2. A relationship between the KD values using either Sepharose-IDA-Me(I1) or PEG-IDA-Me(I1) and the accessible histidine residues on the protein surface can be established. This confirms conservation of the mechanism of protein recognition by the IDA-Me(I1) in the electrical field. Data from experiments with these two polymers used as supports show the validity of this method as an analytical tool for a better design of preparative IMAC or IMAP for a given protein. Moreover, the different KD values for two transition metals, Cu(I1) and Zn(II), can indicate the choice of metal for the preparative IMAC or IMAP method (5).

WHAT ARE THE POTENTIAL APPLICATIONS OF IMA-ELEC?

0.5

Two applications have already been documented We describe here two additional applications.

(5).

1. Followup of Bovine Ribonuclease A Denaturation with 8 M Urea or 6 M Guanidinium Chloride (GuHCI)

0.01 0.0

1 0.2

1 0.4

’ 0.6

’ 0.8

l/(PEG-IDA-Cu(II))

’ 1.0

1.2

mM-’

FIG. 2. Double inverse plot of differential migration (d, - d) versus integrated ligand (L) concentration expressed in mM copper incorporated in the form of PEG 5000-IDA-Cu(I1) for native and denaturated bovine ribonuclease A. Electrophoretic buffer, 0.1 M Trisacetate (pH 7.2); migration time, 7 h at 20 mA. (+) Native ribonuclease A, KD = 22.0 mM; (0) ribonuclease A denaturated by 6 M guanidinium chloride, KD = 3.0 mM; (0) ribonuclease A denaturated by 8 M urea, K. = 7.0 mM.

RNase A was denatured using two well-known denaturing agents, urea and GuHCl, according to the method described (12). The denatured samples, no dialysis, were analyzed by IMA-Elec as illustrated in Figs. lA, LA’, and 1B. The KD values are calculated from Fig. 2. The denatured enzyme showed a systematic increase in affinity for PEG-IDA-Cu(II), with KD values of 7.0 and 3.0 mM for urea-denatured and GuHCl-denatured enzyme, respectively, compared to 22.0 mM for the native enzyme. Further, a mixture of native and denatured RNase gave two bands with different migrations on the gel, as shown in Fig. 1B.

METAL

AFFINITY

This increased affinity of the denatured species for IDA-Cu(I1) can be attributed to unfolding, which will make the histidine residues of the protein more accessible. This is in good agreement with the increased affinities (increased retention) observed for denatured serine proteases (7) or for denatured RNase A in the IMAC system using Sepharose IDA-Cu(I1) (unpublished data). Nevertheless, the differences in the Ko values observed for RNase denatured by urea and that denatured by GuHCl require further discussion. Of these two denaturants, urea mainly ruptures intramolecular hydrogen bonds, while GuHCl additionally induces the protein to dissolve, as GuHCl competes for the proteinbound water molecules. Moreover, comparison of these data with those obtained by fluorescence measurements and/size exclusion chromatography as described in (13) are necessary to validate the method. 2. Study of Glycosylated and Glycated HistidineContaining Proteins Previous reports have indicated a decrease in RNase affinity for immobilized IDA-Cu(I1) when the enzyme is N-glycosylated (5, 7). This was attributed mainly to a steric hindrance effect on the histidine accessibility. Apart from the N- or 0-glycosylation of proteins, the “neoglycoenzymes” are being synthesized for increased enzyme stability (14). The mono- or oligosaccharide

ELECTROPHORESIS

residues are chemically coupled to protein lysyl residues as described in (15). Compared to native enzymes, these neoglycoenzymes are known to differ in their isoelectric points. In this study we have investigated a few histidine-containing enzymes for their histidine accessibility/microenvironment modification resulting from glycation. Figures 3A and 3B show the K. values for IDACu(I1) of subtilisin and three of its glycated derivatives and those for bovine RNase A and three of its glycated derivatives, respectively. We observed a systematic decrease in Ko values following glycation. Moreover, the decrease was more pronounced with increasing sugar moiety size in the case of subtilisin. Further, we have observed decreases in enzyme activity and pl values for the glycated derivatives of subtilisin (Table 3). This concurrent increase in affinity and decrease in enzyme activity may indicate a somewhat different folding of the enzyme, resulting in exposure of the histidine residues (active site residues?). However, modifications in the protein (His ?) microenvironment could induce solvation effects that are similar to those observed in protein denaturation using GuHCl.

CONCLUSION These results with a few protein models clearly indicate the utility of this emerging analytical tool for

J 2

l/(Sepharose-IDA-Cu(II))

mM-l

l(PEG-IDA-Cu(II))

rnT+

FIG. 3. (A) Double inverse plot of differential migration (d, - d) versus integrated ligand (L) concentration expressed in mM copper incorporated in the form of Sepharose GB-IDA-Cu(I1) of subtilisin and its glycated derivatives. Electrophoretic buffer, 0.1 M Tris-acetate (pH 7.2); migration time, 10 h at 20 mA. (0) Native subtilisin, Ko = 4.3 mM; (+) subtilisin glycated with glucose, Ko = 3.4 mM; (0) subtilisin glycated with cellobiose, Ko = 2.4 mM; (0) subtilisin glycated with maltotriose, K n = 1.6 mM. (B) Double inverse plot of differential migration (d, - d) versus integrated ligand (L) concentration expressed in mM copper incorporated in the form of PEG 5000-IDA-Cu(I1) of native and glycated bovine ribonuclease A. Electrophoretic buffer, 0.1 M Tris-acetate (pH 7.2); migration time, 7 h at 20 mA. (m) Native ribonuclease A, Kn = 22.0 mM; (0) the N-glycosylated native ribonuclease B, Ko = 67.0 mM; (+) ribonuclease A glycated with glucose, KD = 1.5 mM; (0) ribonuclease A glycated with cellobiose, KD = 7.5 mM.

NANAK,

102

TABLE Relation Activity

between for Different

IDA-Cu(I1) Glycated KD

Native subtilisin Subtilisin coupled Glucose Cellobiose Maltotriose Note.

ND,

bM)

ABDUL-NOUR,

AND

3 Affinity Subtilisin Ap1

4.30

0

3.40 2.40 1.59

0.2 0.2

REFERENCES and Enzyme Derivatives Residual

activity

(%)

100

with

ND

VIJAYALAKSHMI

50 35 ND

not determined.

1. Horeijsi, 181.

V., and Kocourek,

2. Bog-Hansen, 71.

T. C., and Takeo,

3. Ticha, M., Horeijsi, Acta 534, 58-63.

Methods

K. (1980)

V., and Barthova,

5. Goubran-Botros, H., G., and Vijayalakshmi,

7. Sulkowski, (Burgess,

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34,

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M. A. (1991)

17%

1,67-

Electrophoresis

4. Goubran-Botros, H., and Vijayalakshmi, phoresis 12, 1028-1032.

364. 6. Vijaylakshmi,

protein studies. The potential and possible extension of this technique to molecules other than proteins are evident, and such studies are under way in this laboratory. Moreover, protein-protein and protein-nucleic acid interactions can also be followed by IMA-Elec.

J. (1974)

Biophys. Electro-

Nanak, E., Abdul-Nour, J., Birkenmeir, M. A. (1992) J. Chromatogr. 597, 357-

M. A. (1989)

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E., and Arnold,

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ACKNOWLEDGMENTS The sustained interest of and encouraging discussions with Professors Milan Bier and Jerker Porath (Tucson, AZ) are gratefully acknowledged.

12. Volkin, D. B., and Klibanov, A. M. (1989) in Protein (Creighton, T. E., Ed.), p. 8, IRL Press, Oxford. 13. Herzold, 391.

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