- Email: [email protected]
Immobilized pH gradients: The state of the art
16
trends in analytical chemistry, vol. 5, no. j, 1986
Immobilized pH gradients: the state of the art Pier Giorgio Righetti Milan, Italy
Immobilized pH gradients (IPGs) is a misnomer: to be able to graft a proton onto a polyacrylamide gel is quite a difficult proposition. Nevertheless, by immobilizing in the gel network buffering ions and titrants in given ratios, it is possible to ensure pH constancy in the surrounding liquid elements, i.e. to keep constant the local proton concentration (the pH tout court). Briefly, IPGs represent the latest evolutionary event in isoelectric focusing (IEF)‘, a fractionation technique based on the electrophoretic transport and condensation of amphoteric species (from simple amino acids to large proteins) at their isoelectric point (pl) along a pH gradient. Conventional IEF has been plagued by, among other problems, the instability of pH gradients with time (cathodic drift); IPGs have solved this and most of the drawbacks inherent to IEF in amphoteric buffers (carrier ampholytes). The other spectacular advance of IPGs is their capability of engineering any type of pH gradient (linear, non-linear, step-wise, from extremely shallow to very broad) along the separation axis, thus rendering them potentially able to solve any fractionation problem involving differences in surface charge, no matter how minute, among amphoteric macromolecules. Since I introduced IPGs to TrAC readersz, the technique has expanded considerably: I will attempt to summarize the highlights of this progress and survey potential fields of application. A literature survey I will start this review with a list of fundamental articles in the field of IPGs: they contain practically all the basic developments and the information the readers will be looking for in this new electrophoretic technique: (4 ref. 3: the milestone article, laying the foundations of IPGs; @I refi. 4-8: descriptions of extended pH intervals, spanning 2-6 pH units (computer programs and formulations); ref. 9: a new, acidic pH 3-4 interval; veJ 10: comparison among different focusing techniques in non-amphoteric buffers; (4 refs. 11-15: strategies for optimizing preparative runs (manipulations of gel thickness, pH gradient width, buffering power, ionic strength; 0165-9936/86/$02.00
the discovery of the high loading capacity of ‘soft gels’; retrieval of proteins from Immobiline matrices); (4 refs. 16-18: co-polymerization kinetics of Immobilines into polyacrylamide gels; swelling kinetics of dried IPG gels and stability of Immobilines and pre-cast IPG gels; (e>refs. 19-23: the first two-dimensional (2-D) maps and the use of urea and detergents in IPG systems; (f) refs. 24-26: early reviews in the field of IPGs and 2-D maps; (g>ref. 27: the first (and I hope the last) artefact with the IPG technique; 04 refs. 28 and 29: zymograms and substrate gradients in IPGs. Fractionation of small amphoteric molecules It is well known that IPGs perform wonders in protein fractionations (species differing in isoelectric point by barely dpZ = 0.001 pH unit have been fully resolved)3, but what good will they be to an analytical chemist working with simple molecules? Fear not: if your pet molecule is amphoteric, you might get some mileage out of it. Already with conventional IEF we had demonstrated the possibility of fractionating a small drug, adriamycin, used in liquid tumor therapy, and measuring its physico-chemical parameters, like the degree of aggregation and unknown p&so. With IPGs, the technique has been further expanded; it is possible to separate oligopeptides, barely four to five amino acids in length, and to reveal them inthe gel with general stains for primary amino groups, like ninhydrin, fluorescamine, dansyl chloride, etc. since the background buffers grafted into the gel, unlike carrier ampholytes, are unreactivesi. But recently we have pushed the technique even further: in collaboration with Dr. M. Bier (Tucson, AZ, U.S.A.), we have fractionated the twentyone free amino acids, in a very acidic, pH 3.1-4.1 immobilized gradientsz. In the separation, shown in Fig. 1, there are some quite novel features: (a) by pre-dansylating the amino acids, all the mono-amino, mono-carboxyl species are transformed from ‘poor’ into ‘good’ carrier ampholytes, as the high-pK primary amino group is substituted with a low-pK tertiary amino group; (b) by pre-dansylation, all the derivatives can be detected at the level of barely 2 ng in the gel; (c) the excess dansyl probe does not have to be elimQElsevier
Science Pub1ishersB.V.
*
trends tk analyticalchemistry,
17
vol. 5, no. I, 1986
Fig. 1. Focusing of twenty-one dansyl amino acids in a pH 3. I-4. I Immobiline gel. Conditions: 6 % T [% T = total monomer concentration (acrylamide + bisacrylamide)], 4 %C (%C = grams of cross-linker per 100 g of monomer) acrylamide gel with three times the standard amount of Immobilines (ca. 30 mM buffering ion). Run: 5 h at 1200 V (at equilibrium), 5 W max. at 4°C. Detection byfluorescence. Amino acids from left to right: Asp, Cys, Cys,, Glu, Met, Asn, Gin, Pro, Ser, Gly, Thr, Ala, Leu, Phe, Val, Ile, Trp, Tyr, His, Lys, Arg. They are listed in Table I in order of increasing pl.
inated from the mixture, as it is hydrolyzed to dansyl sulphate, which has a very low pZ and completely different fluorescence (yellow-green acidic band at pZ = 3.34); (d) this is the first example of a quite acidic (pH TABLE I. Calculation of the pKs of the dansyl moieties the pl-pK, values of derivatized amino acids.
from
Data from ref. 32. Amino acid
PI
pK,*
PK,,,**
Dns-OH
3.34 3.43
1.88
_ -
3.48 3.50 3.55
1.92 2.1 2.19
4.77 5.08 -
3.56 3.57 3.58 3.59 3.63 3.67 3.68 3.69 3.70 3.72 3.75 3.76 3.78 3.81 3.88 3.90 8.8
2.28 1.95 2.17 2.02 2.19 2.09 2.35 2.36 2.34 2.16 2.36 2.32 2.20 2.43 1.82 2.16 1.82
5.17 5.10 5.07 5.02 5.09 5.15 5.16 5.10 5.08 5.10 5.15 5.16 5.20 5.19 -
Asp Cys CYs* Glu Met Pro Gin Asn Ser Thr
GlY Leu Ala Phe Be Val Tyr
Trp His Lys
Arg *pK, l
* PK,,,
= dissociation constant of the a carboxyl group in the unreacted amino acid. dissociation constant of the dimethylamino group of = the dansyl derivative (calculated from the pI and PK,).
3.1-4.1) pH gradient which is completely stable against electroendoosmosis. As shown in Table I, the technique can be stretched out even further, and can be used as a physico-chemical probe: we noticed that by arranging the twenty-one dansyl derivatives in order of increasing pZ, most of them were also aligned (except for the ones with ionizable side chains) in order of increasing pK of their carboxyl groups. From the known relation pZ = (pK, + pK.J/2 it was thus possible, from each couple of values, to calculate the unknown pK in the dansyl moiety, which are reported in the last column in Table I. By averaging all these values, the unknown pK was found to be 5.05 + 0.06. Such a well-behaved system had not been predicted by computer simulation of very acidic and very alkaline IPG ranges33. Ampholine-lmmobiline gels I have just stated that the old system of IEF in carrier ampholytes (CA, or Ampholine) should be abandoned in favor of the much advanced IPG technique. Yet recently we have resorted to a melange of the two, a primary, immobilized pH gradient with a superimposed, secondary, CA-pH gradient. We stumbled into that when trying to analyze in IPGs membrane proteins dissolved in 2% Nonidet P-40 (NP-40, a neutral detergent): very few bands focused, the rest being smeared on the gel surface or precipitated at the application point (Fig. 2). We then resorted to an original idea we described years ago: the use of mixed ‘Ampholine-Immobiline’ gel+. Both sample and gel contained 2% NP-40 and 4% Ampholine (pH 4-8) superimposed to a pH 4-8 Immobiline gradient grafted in the matrixss. As
18
trends in analytical
chemistry,
vol. 5, no: I, 1986 f
Fig. 2. IEF of plasma membrane proteins of a capsulated strain of S. cremoris in a pH 4-8 IPG. (A) 3 % T, 4 %C polyacrylamide gel containing 8 M urea and 2% NP-40. Sample: 75 ,ug protein in 30~1 of 2% NP-40 and 10 mM Tris-HCI, pH 8.0. (B) Same as in (A) but with sample and gel containing 4% Ampholine pH 4-8 and no urea. (C) Pattern of focused carrier ampholytes as in (B) visualized by complexing with Pbz+ and precipitating in situ PbS. All gels were focused for 30 000 Vh at 10°C (from ref. 35).
shown in Fig. 2B, the results were astonishing: not only the total solubilization level was increased (from barely 40% in the absence to more than 60% in the presence of CAs) but the bands were all sharply focused and almost no precipitate was present at the application point. As the solubilization mixture was urea-free, it was possible to zymogram a membrane ATP-ase, which seems to consist of two darkbrown bands in Fig. 2C (the fainter bands representing Pb-chelates with the CA chemicals, subsequently precipitated as PbS in the zymogram protocol). The reasons? There are at least two mechanisms involved: (a) CAs form mixed micelles with NP-40, thus producing a zwitterionic detergent, which has a greater solubilizing power on membrane proteins; and (b) the excess free CAs increase the background conductivity of the IPG gel, allowing for quick migration of the proteins to the pl position. This seems to be a quite general mechanism: e.g. when focusing microvillar membrane hydrolases, a compressed pattern was obtained with conventional IEF, a bad smear in IPGs, but an incredible array of sharply focused bands in the mixed CA-IPG technique (Fig. 3)29. Incidentally, CA-IPG gels also allow for proper pH assessments in extremely narrow (0.2-0.3) pH intervals36J7.
Fig. 3. Comparison of bands of enzymatic activity using Gly-ProI-methoxy-2naphthylamine as a substrate for dipeptidyl peptidase IV under different electrophoretic conditions. CA: conventional IEF with carrier ampholytes in the range pH 4-6.5; IPG: immobilized pH gradient in the pH 4-6.5 range; IPG-I % CA: mixed IPGs pH 4-6.5 with 1% Ampholine added; IPG-4% CA: mixed IPGs pH 4-6.5 with 4% Ampholine added. Cathode at the top. Conditions for IPGs: run at 2 W constant power for 12 h at 10°C using 10 mM Glu and 10 mM Lys as anolyte and catholyte, respectively (from ref. 29).
Detection of neutral mutations I have some good news also for clinical chemists: in the vast majority of cases, neutral amino acid substitutions in human proteins go undetected, as there is apparently no charge change altering their electrophoretic mobility. The paucity of neutral variants discovered to date reflects the difficulty of detecting neutral substitutions electrophoretically: as they represent cu. two-third of all possible spot mutations, this means that the estimates of the amount of genetic variation and of the frequency of mutations
19
trends in analytical chemistry, vol. 5, no. I,1986
References 1 P. G. Righetti, Isoelectric Focusing: Theory, Methodology and Applications, Elsevier, Amsterdam, 1983. 2 P. G. Righetti, Trends Anal. Chem., 2 (1983) 193-196. 3 B. Bjellqvist, K. Ek, P. G. Righetti, E. Gianazza, A. Gorg, R. Westermeier and W. Postel, J. Biochem. Biophys. Meth-
Fig. 4. Separation of Hb A and Hb San Diego by IPGs. The gel was 125 X 110 mm, I mm thick and contained 5 % T, 4 %C acrylamide and Immobilines of pK 7.0 and 3.6 in such ratios as to generatea 0.8pH unitspan frompH 6.9 topH 7.7. About8mgof total protein were loaded on the right-hand trench. In the left-hand pocket, 1.5 mg of Hb from a normal adult lysate were applied (from ref. 38).
ods, 6 (1982) 317-339. 4 G. Dossi, F. Celentano, E. Gianazza and P. G. Righetti, J. Biochem. Biophys. Methods, 7 (1983) 123-142. 5 E. Gianazza, G. Dossi, F. Celentano and P. G. Righetti, J. Biochem. Biophys. Methods, 8 (1983) 109-133. 6 E. Gianazza, F. Celentano, G. Dossi, B. Bjellqvist and P. G. Righetti, Electrophoresis, 5 (1984) 88-97. 7 E. Gianazza, S. Astrua-Testori and P. G. Righetti, Electrophoresis, 6 (1985) 113-117. 8 E. Gianazza, P. Giacon, B. Sahlin and P. G. Righetti, Electrophoresis, 6 (1985) 53-56. 9 P. G. Righetti, E. Gianazza and F. Celentano, J. Chromatogr., 356 (1986) in press. 10 P. G. Righetti and E. Gianazza, J. Chromatogr., 334 (1985) 71-82. 11 K. Ek, B. Bjellqvist and P. G. Righetti, J. Biochem. Biophys. Methods, 8 (1983) 134-155. 12 C. Gelfi and P. G. Righetti, J. Biochem. Biophys. Methods, 8 (1983) 156-171. 13 P. G. Righetti and C. Gelfi, J. Biochem. Biophys. Methods, 9 (1984) 103-119. 14 P. Casero, C. Gelfi and P. G. Righetti, Electrophoresis, 6 (1985) 59-69. 15 R. Bartels and L. Bock, in V. Neuhoff (Editor), Electrophoresis ‘84, Verlag Chemie, Weinheim, 1984, pp. 103-106. 16 P. G. Righetti, K. Ek and B. Bjellqvist, J. Chromatogr., 291 (1984) 31-42. 17 C. Gelfi and P. G. Righetti, Electrophoresis, 5 (1984) 2.57-262.
in human populations have been grossly underestimated. With IPGs, things are rapidly changing: as shown in Fig. 4, Hb San Diego (a neutral, Val + Met substitution) could be amply resolved from its nearest neighbor, Hb A (normal human adult hemoglobin)3s. Note that conventional IEF or chromatographic techniques could not tell the two species apart. We have also been able to resolve a mutant of a fetal (F) globin chain (y) bearing a substitution in residue No. 75 of y-globin: a threonine instead of an isoleucine (F-Sardinia) 39. Even tougher than that, we have been able to separate two naturally occurring fetal hemoglobin populations: a tetramer Ay vs. a tetramer Gy, the latter bearing a Gly in residue No. 136 instead of an Ala39. Note that all these separations have been performed in native hemoglobin tetramers, without resorting to preparation of hemefree globin chains or digestion with proteases and fingerprinting. Clearly the horizon of clinical chemistry is rapidly expanding: a novel case of the Big Bang?
20 E. Gianazza, F. Artoni and P. G. Righetti, Electrophoresis,
Conclusions In my last article in TrAC2 I urged you to throw your dice, like Caesar, and cross the Rubicon. I hear a lot of you were drowned in the crossing (how could you, since this summer it has not been raining for five months, and the Rubicon is but a trickle of water?). Well I hope the present progress report will be your life-saver. A message to all those drowned scientists: come back from your ultramundane life and try again! I know you will succeed.
29 P. K. Sihna and P. G. Righetti, J. Biochem. Biophys. Methods, 12 (1986) in press. 30 P. G. Righetti, M. Menozzi, E. Gianazza and L. Valentini, FEBS Letters, 101(1979) 51-55. 31 E. Gianazza, F. Chillemi, M. Duranti and P. G. Righetti, J.
18 Pietta, P. G., E. Pocaterra, A. Fiorino, E. Gianazza and P. G. Righetti, Electrophoresis, 6 (1985) 162-170. 19 R. Westermeier, W. Postel, J. Weser and A. Gorg, J. Biothem. Biophys. Methods, 8 (1983) 321-330. 4 (1983) 321-326.
21 E. Gianazza, A. Frigerio, A. Tagliabue Electrophoresis,
22 E. ti, 23 E. ti, 24 P.
and P. G. Righetti,
5 (1984) 209-216.
Gianazza, P. Giacon, S. Astrua-Testori Electrophoresis,
Gianazza, S. Astrua-Testori, Electrophoresis,
and P. G. Righet-
6 (1985) 326-331.
P. Giacon and P. G. Righet-
6 (1985) 332-339.
G. Righetti, E. Gianazza and B. Bjellqvist, J. Biochem. Biophys. Methods, 8 (1983) 89-108. 25 P. G. Righetti, J. Chromatogr., 300 (1984) 165-223. 26 P. G. Righetti, E. Gianazza and C. Gelfi, in V. Neuhoff (Editor), Electrophoresis ‘84, Verlag Chemie, Weinheim, 1984, pp. 29-48. 27 P. G. Righetti, M. Delpech, F. Moisand, J. Kruh and D. Labie, Electrophoresis, 4 (1983) 393-398. 28 A. G&g, W. Postel and P. Johann, J. Biochem. Biophys. Methods, 10 (1985) 341-350.
Biochem. Biophys. Methods, 8 (1983) 339-351.
32 A. Bianchi-Bosisio, Electrophoresis,
P. G. Righetti, N. B. Egen and M. Bier,
7 (1986) in press.
20
trendsin analyticalchemistry,vol. 5, no.‘l, 1986 T
33 R. A. Mosher, M. Bier and P. G. Righetti, Electrophoresis, 7 (1986) in press. 34 P. G. Righetti and C. Macelloni, J. Biochem. Biophys. Methods, 6 (1982) 1-15. 35 M. A. Rimpilainen and P. G. Righetti, Electrophoresis, 6 (1985) 419-422. 36 P. G. Righetti, A. Morelli and C. Gelfi, J. Chromatogr., (1986) in press. 37 C. Gelfi, A. Morelli, E. Rovida and P. G. Righetti, J. Biothem. Biophys. Methods, (1986) in press. 38 J. Rochette, P. G. Righetti, A. Bianchi-Bosisio, F. Vertongen, G. Schneck, J. P. Boissel, D. Labie and H. Wajcman, J. Chromatogr., 285 (1984) 143-152.
39 M. Manta, G. Cossu, A. Bianchi-Bosisio, E. Gianazza and P. G. Righetti, Nature (London), (1986) in press.
Pier Giorgio Righetti received his Ph.D. in Chemistry at the University of Pavia. He is currently Professor of Biochemistry at the Faculty of Pharmacy at the University of Milan, Italy. He is the author of several books on electrophoresis and has been supported, for the development of IPGs, by two five-year grants from Consiglio Nazionale delle Ricerche (Roma), Progetti Finalizzati ‘Salute dell’Uomo’ and ‘Chimica Fine’.
Polarographic and voltammetric techniques and their application to the determination of vitamins and coenzymes J. P. Hart London, U.K. The application of modern polarographic and voltammetric techniques to the analysis of vitamins and coenzymes overcomes many of the problems of analysing these compounds in complex matrices such as foods, pharmaceuticals and biological fluids.
Although polarography had great potential in microanalysis, it is only recently, with improvement in selectivity and sensitivity, that this potential is being realised. In polarography, and other variants of voltammetry, two types of current are produced: the capacity current, arising from the flow of current required to charge a double-layer which forms at the surface of the working electrode, and the faradaic current which is the result of electrons being transferred to, or removed from, the electroactive species. It is the latter which is required and which modern techniques discriminate. Since the techniques have been reviewed in detail recently (Table I), I will consider in the present survey some applications to the analysis of vitamins and coenzymes. Applications Fat-soluble vitamins
The fat-soluble vitamins A and E are not readily determined by polarographic methods because they do not possess a functional group which can be easily reduced whereas their oxidation occurs at potentials which are not accessible with mercury electrodes (mercury oxidises at potentials more positive than about +0.4 V). However, carbon electrodes extend 0165-9936/86/$02.00.
the accessible range of positive potential to about +2.0 V, and several studies have clearly demonstrated the utility of these electrodes for the voltammetric analysis of compounds of the vitamin A and E groups using their oxidation peaks. For example, Atuma and co-worker@ developed a carbon paste electrode which could be used in a variety of organic solvents without dissolution; a new electrode could be obtained by simply slicing a small piece off the end of the electrode with a wire. The oxidation potentials obtained for some compounds of the vitamin A and E groups are shown in Table II. With this carbon paste electrodes it was possible to determine vitamin E (tocopherols) in pharmaceuti-
TABLE I. Some polarographic and voltammetric techniques and their limits of detection which have been used for the analysis of vitamins and coenzymes. Technique Classical direct current (d.c.) polarography (original polarographic technique) Linear sweep voltammetry Alternating current (a.c.) polarography Normal pulse polarographyi voltammetry Differential pulse polarographyi voltammetry Stripping voltammetry Liquid chromatography with electrochemical detection (LC-ED)
Limit of detection (M)
Reference
-5.10-5 -10-e
l-3 l-3
-10-G
l-3
10-6-10-7 lo-‘-lo-* -10-10
-10-U
l-3 l-3 2,3
1,394
0 Elsevier Science Publishers B.V.
trends in analytical chemistry, vol. 5, no. j, 1986
Immobilized pH gradients: the state of the art Pier Giorgio Righetti Milan, Italy
Immobilized pH gradients (IPGs) is a misnomer: to be able to graft a proton onto a polyacrylamide gel is quite a difficult proposition. Nevertheless, by immobilizing in the gel network buffering ions and titrants in given ratios, it is possible to ensure pH constancy in the surrounding liquid elements, i.e. to keep constant the local proton concentration (the pH tout court). Briefly, IPGs represent the latest evolutionary event in isoelectric focusing (IEF)‘, a fractionation technique based on the electrophoretic transport and condensation of amphoteric species (from simple amino acids to large proteins) at their isoelectric point (pl) along a pH gradient. Conventional IEF has been plagued by, among other problems, the instability of pH gradients with time (cathodic drift); IPGs have solved this and most of the drawbacks inherent to IEF in amphoteric buffers (carrier ampholytes). The other spectacular advance of IPGs is their capability of engineering any type of pH gradient (linear, non-linear, step-wise, from extremely shallow to very broad) along the separation axis, thus rendering them potentially able to solve any fractionation problem involving differences in surface charge, no matter how minute, among amphoteric macromolecules. Since I introduced IPGs to TrAC readersz, the technique has expanded considerably: I will attempt to summarize the highlights of this progress and survey potential fields of application. A literature survey I will start this review with a list of fundamental articles in the field of IPGs: they contain practically all the basic developments and the information the readers will be looking for in this new electrophoretic technique: (4 ref. 3: the milestone article, laying the foundations of IPGs; @I refi. 4-8: descriptions of extended pH intervals, spanning 2-6 pH units (computer programs and formulations); ref. 9: a new, acidic pH 3-4 interval; veJ 10: comparison among different focusing techniques in non-amphoteric buffers; (4 refs. 11-15: strategies for optimizing preparative runs (manipulations of gel thickness, pH gradient width, buffering power, ionic strength; 0165-9936/86/$02.00
the discovery of the high loading capacity of ‘soft gels’; retrieval of proteins from Immobiline matrices); (4 refs. 16-18: co-polymerization kinetics of Immobilines into polyacrylamide gels; swelling kinetics of dried IPG gels and stability of Immobilines and pre-cast IPG gels; (e>refs. 19-23: the first two-dimensional (2-D) maps and the use of urea and detergents in IPG systems; (f) refs. 24-26: early reviews in the field of IPGs and 2-D maps; (g>ref. 27: the first (and I hope the last) artefact with the IPG technique; 04 refs. 28 and 29: zymograms and substrate gradients in IPGs. Fractionation of small amphoteric molecules It is well known that IPGs perform wonders in protein fractionations (species differing in isoelectric point by barely dpZ = 0.001 pH unit have been fully resolved)3, but what good will they be to an analytical chemist working with simple molecules? Fear not: if your pet molecule is amphoteric, you might get some mileage out of it. Already with conventional IEF we had demonstrated the possibility of fractionating a small drug, adriamycin, used in liquid tumor therapy, and measuring its physico-chemical parameters, like the degree of aggregation and unknown p&so. With IPGs, the technique has been further expanded; it is possible to separate oligopeptides, barely four to five amino acids in length, and to reveal them inthe gel with general stains for primary amino groups, like ninhydrin, fluorescamine, dansyl chloride, etc. since the background buffers grafted into the gel, unlike carrier ampholytes, are unreactivesi. But recently we have pushed the technique even further: in collaboration with Dr. M. Bier (Tucson, AZ, U.S.A.), we have fractionated the twentyone free amino acids, in a very acidic, pH 3.1-4.1 immobilized gradientsz. In the separation, shown in Fig. 1, there are some quite novel features: (a) by pre-dansylating the amino acids, all the mono-amino, mono-carboxyl species are transformed from ‘poor’ into ‘good’ carrier ampholytes, as the high-pK primary amino group is substituted with a low-pK tertiary amino group; (b) by pre-dansylation, all the derivatives can be detected at the level of barely 2 ng in the gel; (c) the excess dansyl probe does not have to be elimQElsevier
Science Pub1ishersB.V.
*
trends tk analyticalchemistry,
17
vol. 5, no. I, 1986
Fig. 1. Focusing of twenty-one dansyl amino acids in a pH 3. I-4. I Immobiline gel. Conditions: 6 % T [% T = total monomer concentration (acrylamide + bisacrylamide)], 4 %C (%C = grams of cross-linker per 100 g of monomer) acrylamide gel with three times the standard amount of Immobilines (ca. 30 mM buffering ion). Run: 5 h at 1200 V (at equilibrium), 5 W max. at 4°C. Detection byfluorescence. Amino acids from left to right: Asp, Cys, Cys,, Glu, Met, Asn, Gin, Pro, Ser, Gly, Thr, Ala, Leu, Phe, Val, Ile, Trp, Tyr, His, Lys, Arg. They are listed in Table I in order of increasing pl.
inated from the mixture, as it is hydrolyzed to dansyl sulphate, which has a very low pZ and completely different fluorescence (yellow-green acidic band at pZ = 3.34); (d) this is the first example of a quite acidic (pH TABLE I. Calculation of the pKs of the dansyl moieties the pl-pK, values of derivatized amino acids.
from
Data from ref. 32. Amino acid
PI
pK,*
PK,,,**
Dns-OH
3.34 3.43
1.88
_ -
3.48 3.50 3.55
1.92 2.1 2.19
4.77 5.08 -
3.56 3.57 3.58 3.59 3.63 3.67 3.68 3.69 3.70 3.72 3.75 3.76 3.78 3.81 3.88 3.90 8.8
2.28 1.95 2.17 2.02 2.19 2.09 2.35 2.36 2.34 2.16 2.36 2.32 2.20 2.43 1.82 2.16 1.82
5.17 5.10 5.07 5.02 5.09 5.15 5.16 5.10 5.08 5.10 5.15 5.16 5.20 5.19 -
Asp Cys CYs* Glu Met Pro Gin Asn Ser Thr
GlY Leu Ala Phe Be Val Tyr
Trp His Lys
Arg *pK, l
* PK,,,
= dissociation constant of the a carboxyl group in the unreacted amino acid. dissociation constant of the dimethylamino group of = the dansyl derivative (calculated from the pI and PK,).
3.1-4.1) pH gradient which is completely stable against electroendoosmosis. As shown in Table I, the technique can be stretched out even further, and can be used as a physico-chemical probe: we noticed that by arranging the twenty-one dansyl derivatives in order of increasing pZ, most of them were also aligned (except for the ones with ionizable side chains) in order of increasing pK of their carboxyl groups. From the known relation pZ = (pK, + pK.J/2 it was thus possible, from each couple of values, to calculate the unknown pK in the dansyl moiety, which are reported in the last column in Table I. By averaging all these values, the unknown pK was found to be 5.05 + 0.06. Such a well-behaved system had not been predicted by computer simulation of very acidic and very alkaline IPG ranges33. Ampholine-lmmobiline gels I have just stated that the old system of IEF in carrier ampholytes (CA, or Ampholine) should be abandoned in favor of the much advanced IPG technique. Yet recently we have resorted to a melange of the two, a primary, immobilized pH gradient with a superimposed, secondary, CA-pH gradient. We stumbled into that when trying to analyze in IPGs membrane proteins dissolved in 2% Nonidet P-40 (NP-40, a neutral detergent): very few bands focused, the rest being smeared on the gel surface or precipitated at the application point (Fig. 2). We then resorted to an original idea we described years ago: the use of mixed ‘Ampholine-Immobiline’ gel+. Both sample and gel contained 2% NP-40 and 4% Ampholine (pH 4-8) superimposed to a pH 4-8 Immobiline gradient grafted in the matrixss. As
18
trends in analytical
chemistry,
vol. 5, no: I, 1986 f
Fig. 2. IEF of plasma membrane proteins of a capsulated strain of S. cremoris in a pH 4-8 IPG. (A) 3 % T, 4 %C polyacrylamide gel containing 8 M urea and 2% NP-40. Sample: 75 ,ug protein in 30~1 of 2% NP-40 and 10 mM Tris-HCI, pH 8.0. (B) Same as in (A) but with sample and gel containing 4% Ampholine pH 4-8 and no urea. (C) Pattern of focused carrier ampholytes as in (B) visualized by complexing with Pbz+ and precipitating in situ PbS. All gels were focused for 30 000 Vh at 10°C (from ref. 35).
shown in Fig. 2B, the results were astonishing: not only the total solubilization level was increased (from barely 40% in the absence to more than 60% in the presence of CAs) but the bands were all sharply focused and almost no precipitate was present at the application point. As the solubilization mixture was urea-free, it was possible to zymogram a membrane ATP-ase, which seems to consist of two darkbrown bands in Fig. 2C (the fainter bands representing Pb-chelates with the CA chemicals, subsequently precipitated as PbS in the zymogram protocol). The reasons? There are at least two mechanisms involved: (a) CAs form mixed micelles with NP-40, thus producing a zwitterionic detergent, which has a greater solubilizing power on membrane proteins; and (b) the excess free CAs increase the background conductivity of the IPG gel, allowing for quick migration of the proteins to the pl position. This seems to be a quite general mechanism: e.g. when focusing microvillar membrane hydrolases, a compressed pattern was obtained with conventional IEF, a bad smear in IPGs, but an incredible array of sharply focused bands in the mixed CA-IPG technique (Fig. 3)29. Incidentally, CA-IPG gels also allow for proper pH assessments in extremely narrow (0.2-0.3) pH intervals36J7.
Fig. 3. Comparison of bands of enzymatic activity using Gly-ProI-methoxy-2naphthylamine as a substrate for dipeptidyl peptidase IV under different electrophoretic conditions. CA: conventional IEF with carrier ampholytes in the range pH 4-6.5; IPG: immobilized pH gradient in the pH 4-6.5 range; IPG-I % CA: mixed IPGs pH 4-6.5 with 1% Ampholine added; IPG-4% CA: mixed IPGs pH 4-6.5 with 4% Ampholine added. Cathode at the top. Conditions for IPGs: run at 2 W constant power for 12 h at 10°C using 10 mM Glu and 10 mM Lys as anolyte and catholyte, respectively (from ref. 29).
Detection of neutral mutations I have some good news also for clinical chemists: in the vast majority of cases, neutral amino acid substitutions in human proteins go undetected, as there is apparently no charge change altering their electrophoretic mobility. The paucity of neutral variants discovered to date reflects the difficulty of detecting neutral substitutions electrophoretically: as they represent cu. two-third of all possible spot mutations, this means that the estimates of the amount of genetic variation and of the frequency of mutations
19
trends in analytical chemistry, vol. 5, no. I,1986
References 1 P. G. Righetti, Isoelectric Focusing: Theory, Methodology and Applications, Elsevier, Amsterdam, 1983. 2 P. G. Righetti, Trends Anal. Chem., 2 (1983) 193-196. 3 B. Bjellqvist, K. Ek, P. G. Righetti, E. Gianazza, A. Gorg, R. Westermeier and W. Postel, J. Biochem. Biophys. Meth-
Fig. 4. Separation of Hb A and Hb San Diego by IPGs. The gel was 125 X 110 mm, I mm thick and contained 5 % T, 4 %C acrylamide and Immobilines of pK 7.0 and 3.6 in such ratios as to generatea 0.8pH unitspan frompH 6.9 topH 7.7. About8mgof total protein were loaded on the right-hand trench. In the left-hand pocket, 1.5 mg of Hb from a normal adult lysate were applied (from ref. 38).
ods, 6 (1982) 317-339. 4 G. Dossi, F. Celentano, E. Gianazza and P. G. Righetti, J. Biochem. Biophys. Methods, 7 (1983) 123-142. 5 E. Gianazza, G. Dossi, F. Celentano and P. G. Righetti, J. Biochem. Biophys. Methods, 8 (1983) 109-133. 6 E. Gianazza, F. Celentano, G. Dossi, B. Bjellqvist and P. G. Righetti, Electrophoresis, 5 (1984) 88-97. 7 E. Gianazza, S. Astrua-Testori and P. G. Righetti, Electrophoresis, 6 (1985) 113-117. 8 E. Gianazza, P. Giacon, B. Sahlin and P. G. Righetti, Electrophoresis, 6 (1985) 53-56. 9 P. G. Righetti, E. Gianazza and F. Celentano, J. Chromatogr., 356 (1986) in press. 10 P. G. Righetti and E. Gianazza, J. Chromatogr., 334 (1985) 71-82. 11 K. Ek, B. Bjellqvist and P. G. Righetti, J. Biochem. Biophys. Methods, 8 (1983) 134-155. 12 C. Gelfi and P. G. Righetti, J. Biochem. Biophys. Methods, 8 (1983) 156-171. 13 P. G. Righetti and C. Gelfi, J. Biochem. Biophys. Methods, 9 (1984) 103-119. 14 P. Casero, C. Gelfi and P. G. Righetti, Electrophoresis, 6 (1985) 59-69. 15 R. Bartels and L. Bock, in V. Neuhoff (Editor), Electrophoresis ‘84, Verlag Chemie, Weinheim, 1984, pp. 103-106. 16 P. G. Righetti, K. Ek and B. Bjellqvist, J. Chromatogr., 291 (1984) 31-42. 17 C. Gelfi and P. G. Righetti, Electrophoresis, 5 (1984) 2.57-262.
in human populations have been grossly underestimated. With IPGs, things are rapidly changing: as shown in Fig. 4, Hb San Diego (a neutral, Val + Met substitution) could be amply resolved from its nearest neighbor, Hb A (normal human adult hemoglobin)3s. Note that conventional IEF or chromatographic techniques could not tell the two species apart. We have also been able to resolve a mutant of a fetal (F) globin chain (y) bearing a substitution in residue No. 75 of y-globin: a threonine instead of an isoleucine (F-Sardinia) 39. Even tougher than that, we have been able to separate two naturally occurring fetal hemoglobin populations: a tetramer Ay vs. a tetramer Gy, the latter bearing a Gly in residue No. 136 instead of an Ala39. Note that all these separations have been performed in native hemoglobin tetramers, without resorting to preparation of hemefree globin chains or digestion with proteases and fingerprinting. Clearly the horizon of clinical chemistry is rapidly expanding: a novel case of the Big Bang?
20 E. Gianazza, F. Artoni and P. G. Righetti, Electrophoresis,
Conclusions In my last article in TrAC2 I urged you to throw your dice, like Caesar, and cross the Rubicon. I hear a lot of you were drowned in the crossing (how could you, since this summer it has not been raining for five months, and the Rubicon is but a trickle of water?). Well I hope the present progress report will be your life-saver. A message to all those drowned scientists: come back from your ultramundane life and try again! I know you will succeed.
29 P. K. Sihna and P. G. Righetti, J. Biochem. Biophys. Methods, 12 (1986) in press. 30 P. G. Righetti, M. Menozzi, E. Gianazza and L. Valentini, FEBS Letters, 101(1979) 51-55. 31 E. Gianazza, F. Chillemi, M. Duranti and P. G. Righetti, J.
18 Pietta, P. G., E. Pocaterra, A. Fiorino, E. Gianazza and P. G. Righetti, Electrophoresis, 6 (1985) 162-170. 19 R. Westermeier, W. Postel, J. Weser and A. Gorg, J. Biothem. Biophys. Methods, 8 (1983) 321-330. 4 (1983) 321-326.
21 E. Gianazza, A. Frigerio, A. Tagliabue Electrophoresis,
22 E. ti, 23 E. ti, 24 P.
and P. G. Righetti,
5 (1984) 209-216.
Gianazza, P. Giacon, S. Astrua-Testori Electrophoresis,
Gianazza, S. Astrua-Testori, Electrophoresis,
and P. G. Righet-
6 (1985) 326-331.
P. Giacon and P. G. Righet-
6 (1985) 332-339.
G. Righetti, E. Gianazza and B. Bjellqvist, J. Biochem. Biophys. Methods, 8 (1983) 89-108. 25 P. G. Righetti, J. Chromatogr., 300 (1984) 165-223. 26 P. G. Righetti, E. Gianazza and C. Gelfi, in V. Neuhoff (Editor), Electrophoresis ‘84, Verlag Chemie, Weinheim, 1984, pp. 29-48. 27 P. G. Righetti, M. Delpech, F. Moisand, J. Kruh and D. Labie, Electrophoresis, 4 (1983) 393-398. 28 A. G&g, W. Postel and P. Johann, J. Biochem. Biophys. Methods, 10 (1985) 341-350.
Biochem. Biophys. Methods, 8 (1983) 339-351.
32 A. Bianchi-Bosisio, Electrophoresis,
P. G. Righetti, N. B. Egen and M. Bier,
7 (1986) in press.
20
trendsin analyticalchemistry,vol. 5, no.‘l, 1986 T
33 R. A. Mosher, M. Bier and P. G. Righetti, Electrophoresis, 7 (1986) in press. 34 P. G. Righetti and C. Macelloni, J. Biochem. Biophys. Methods, 6 (1982) 1-15. 35 M. A. Rimpilainen and P. G. Righetti, Electrophoresis, 6 (1985) 419-422. 36 P. G. Righetti, A. Morelli and C. Gelfi, J. Chromatogr., (1986) in press. 37 C. Gelfi, A. Morelli, E. Rovida and P. G. Righetti, J. Biothem. Biophys. Methods, (1986) in press. 38 J. Rochette, P. G. Righetti, A. Bianchi-Bosisio, F. Vertongen, G. Schneck, J. P. Boissel, D. Labie and H. Wajcman, J. Chromatogr., 285 (1984) 143-152.
39 M. Manta, G. Cossu, A. Bianchi-Bosisio, E. Gianazza and P. G. Righetti, Nature (London), (1986) in press.
Pier Giorgio Righetti received his Ph.D. in Chemistry at the University of Pavia. He is currently Professor of Biochemistry at the Faculty of Pharmacy at the University of Milan, Italy. He is the author of several books on electrophoresis and has been supported, for the development of IPGs, by two five-year grants from Consiglio Nazionale delle Ricerche (Roma), Progetti Finalizzati ‘Salute dell’Uomo’ and ‘Chimica Fine’.
Polarographic and voltammetric techniques and their application to the determination of vitamins and coenzymes J. P. Hart London, U.K. The application of modern polarographic and voltammetric techniques to the analysis of vitamins and coenzymes overcomes many of the problems of analysing these compounds in complex matrices such as foods, pharmaceuticals and biological fluids.
Although polarography had great potential in microanalysis, it is only recently, with improvement in selectivity and sensitivity, that this potential is being realised. In polarography, and other variants of voltammetry, two types of current are produced: the capacity current, arising from the flow of current required to charge a double-layer which forms at the surface of the working electrode, and the faradaic current which is the result of electrons being transferred to, or removed from, the electroactive species. It is the latter which is required and which modern techniques discriminate. Since the techniques have been reviewed in detail recently (Table I), I will consider in the present survey some applications to the analysis of vitamins and coenzymes. Applications Fat-soluble vitamins
The fat-soluble vitamins A and E are not readily determined by polarographic methods because they do not possess a functional group which can be easily reduced whereas their oxidation occurs at potentials which are not accessible with mercury electrodes (mercury oxidises at potentials more positive than about +0.4 V). However, carbon electrodes extend 0165-9936/86/$02.00.
the accessible range of positive potential to about +2.0 V, and several studies have clearly demonstrated the utility of these electrodes for the voltammetric analysis of compounds of the vitamin A and E groups using their oxidation peaks. For example, Atuma and co-worker@ developed a carbon paste electrode which could be used in a variety of organic solvents without dissolution; a new electrode could be obtained by simply slicing a small piece off the end of the electrode with a wire. The oxidation potentials obtained for some compounds of the vitamin A and E groups are shown in Table II. With this carbon paste electrodes it was possible to determine vitamin E (tocopherols) in pharmaceuti-
TABLE I. Some polarographic and voltammetric techniques and their limits of detection which have been used for the analysis of vitamins and coenzymes. Technique Classical direct current (d.c.) polarography (original polarographic technique) Linear sweep voltammetry Alternating current (a.c.) polarography Normal pulse polarographyi voltammetry Differential pulse polarographyi voltammetry Stripping voltammetry Liquid chromatography with electrochemical detection (LC-ED)
Limit of detection (M)
Reference
-5.10-5 -10-e
l-3 l-3
-10-G
l-3
10-6-10-7 lo-‘-lo-* -10-10
-10-U
l-3 l-3 2,3
1,394
0 Elsevier Science Publishers B.V.