The Alpher, Bethe, Gamow of isoelectric focusing, .The Alpher, Bethe, Gamow of isoelectric focusing,

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Text of The Alpher, Bethe, Gamow of isoelectric focusing, .The Alpher, Bethe, Gamow of isoelectric focusing,

  • Pier Giorgio Righetti

    Politecnico di Milano,Department of Chemistry,Materials and Engineering ChemistryGiulio Natta,Milano, Italy

    Received July 20, 2005Revised September 7, 2005Accepted September 8, 2005


    The Alpher, Bethe, Gamow of isoelectricfocusing, the alpha-Centaury ofelectrokinetic methodologies. Part I

    The birth and evolution of IEF in conventional carrier ampholyte buffers is reviewedhere, from a shaky start during World War II, via desperate attempts of Svensson tocreate pH gradients by stationary electrolysis of salts, to the development of the IEFtheory and the solution of the steady-state equation. The remarkable synthetic processof Ampholines, as ingeniously devised by Vesterberg, is additionally recalled, with athorough description of the fundamental properties of these amphoteric buffers,creating and maintaining the pH gradient under strong electric fields. The review endswith a mention of the major contributions of B. J. Radola to this field, namely analyticaland preparative IEF in granulated Sephadex layers and the development of ultrathinIEF, in polyacrylamide gels as thin as 20100 mm. The latter technique paved the way toDNA sequencing gels and to CZE. The symptoms of decay are here presented throughthe simulations of Mosher and Thormann, clearly indicating an isotachophoreticmechanism for pH gradient decay with time. The decay of IEF was the birth of IPGs.

    Le donne, i cavalier, larme, gli amorile cortesie, laudaci imprese io canto,che furo al tempo che passaro i MoridAfrica il mare, e in Francia nocquer tanto. . .

    Ludovico Ariosto, Orlando Furioso, Ferrara, 1532

    Keywords: Buffering power / Carrier ampholytes / Isoelectric focusing / Sephadexbeds / Ultrathin layer gels DOI 10.1002/elps.200500525

    1 Introduction

    I have always been puzzled by the lack, in the scientificliterature, of a kind of heroic perspective of some of themajor discoveries that have given impulse to the scienceof the epoch in which they were reported and haveshaped the future for quite a few decades afterwards.Such examples abound in the humanities, i.e. in thosebranches of learning connected with human thought andrelations, as distinguished from the sciences, such as lit-erature, philosophy, fine arts, history, and the like. Withoutdisturbing Homer, with the heroic epics of Iliad and

    Odyssey, one could recall here a number of sagas thathave spurred our imagination, such as The Quest of theHoly Graal [1], The Nibelungenlied [2], The VinlandSagas [3], The Buccaneers of America [4], to name justa few. Even the four voyages of Christopher Columbusbecame a saga [5], as witnessed by the some50 000 books written about him and his deeds. There is nosuch counterpart in the scientific field. True, the mostimportant discoveries and their discoverers are oftengranted the Nobel Laurel [6], but rarely the accounts oftheir deeds are sculptured in the Hall of Fame andbecome a legend.

    I am glad to be able to exploit this Special Issue ofElectrophoresis, in honor of Professor B. J. Radola, torecount the saga of IEF, the bright star that, like thea-Centaury, one of the brightest stars close to our solarsystem, illuminated the scenario of electrokinetic meth-odology since the early 70s and is not likely to set belowthe horizon even in todays world. As one of the veterans

    Correspondence: Professor Pier G. Righetti, Politecnico di Milano,Department of Chemistry, Materials and Engineering Chemistry Giu-lio Natta, Via Mancinelli 7, I-20131 Milano, ItalyE-mail: piergiorgio.righetti@polimi.itFax: 139-02-23993080

    Abbreviations: CA, carrier ampholyte; Hb, hemoglobin

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    in the field, I think I should be able to offer a vivid scenarioof its development, for the young generation who has nothad a chance to follow the long march towards thesteady state and beyond. As luck goes, most of theaccounts scattered in the literature have been written bypeople who have not had much to do with the develop-ment of the technique, and hardly know its intricacies.Their accounts most often have been obtained by lootingthe work of those who had been there since the very start.

    2 The steady state

    The long march towards the steady state, the trueessence of IEF, was studded with obstacles disseminatedalong the path of young Harry Svensson (who in 1968changed his name to Rilbe). That winter of 1942 musthave been the most dreadful one of his life. As a youngpupil, under the iron fist of Dom Arne Tiselius, he wasforced to spend endless days and nights trying to make adream of his boss come true, namely the creation of a pHgradient by stationary electrolysis of a salt solution.Among the innumerable problems encountered, onebecame immediately apparent: the ionic constituentswere completely swept to the opposite-charge electrode.Although equilibrium between ion transport and back dif-fusion ensued, at neutral pH, midway in the apparatuswhere a region of water almost completely devoid of ionsformed, ohmic resistance increased enormously, with theresult that the liquid almost boiled at this point. No usefulpH gradient could obviously ensue. When performing,under convection-free conditions, electrolysis of sodiumsulfate, only two portions of a pH course could be gener-ated: a pH 1.72.6 in the region of free H2SO4 and apH 11.512.3 in the zone of free NaOH [7], practicallyuseless for most protein/peptide separations.

    Luckless Harry was chained to the bench adding saltdropwise from a burette, trying to fight the conductivityminimum. Add to this the fact that it was the peak of WorldWar II: Norway and Denmark were occupied by Germany;Finland was oppressed by neighboring Russia and Swe-den ducked low under a declaration of neutrality, butnevertheless under quite miserable conditions. Harrydreamed of escaping this agonizing life and paltry winter,repairing to England, to be drafted in the army and sent tothe African desert to fight the ItalianGerman coalition. Atleast he would have enjoyed sunny days and warmweather! As luck would have it, Tiselius relaxed his irongrip and let Harry present his Ph.D. thesis in 1946 (it wason Electrophoresis by the Moving Boundary Method)[8]. This was no ordinary thesis, mind you, for Harrysmentors were The Svedberg, a 1926 Nobel laureate andTiselius, a future Nobelist (1948).

    Hapless Harry must have been obsessed for the re-mainder of his life by this idea of creating pH gradients.Already in 1956 he dreamed of a hypothetical biproticampholyte having two intrinsic pKa values both equal to7.0. If it were ever to exist, a solution of it in pure waterwould be a neutral buffer containing one-fourth of theamount in the cationic and one-fourth in the anionicform. A superb carrier ampholyte (CA), indeed, whichwould offer a substantial conductivity where it was mostneeded, i.e. in that terrible conductivity Grand Canyonlocated at pH 7, but would still be isoelectric and immo-bile in the electric field [9]. Although such a superbuffercould not possibly exist, 3 years later, during a leave ofabsence at Caltech, chez Linus Pauling, Svensson founda real chemical that came rather close to that: histidyl-histidine, with pKa values at 6.8 and 7.8, thus isoelectricat pH 7.3. It was used with hemoglobins (Hb) in the firstexperiments [10].

    Once back home, as a freelance at the Karolinska Insti-tute, Harry worked hard on the theory of IEF and laiddown its theoretical foundations in a couple of, by now,classic papers [11, 12]. His secret battle cry as he wagedhis underground guerrilla warfare against the Maestrosmammoth instrumentation: no more moving bound-aries! He had a point. It had been foolish to try to createstable and stationary pH gradients in the presence of anelectric field with nonamphoteric compounds. These willvacate the grounds and leave an empty trail in their wakewith no soldiers to guard the battlefield. All buffers had tobe amphoteric and, in addition, they had to have decentbuffering power as ensured by not too large DpKa values.Only in this way would the zones of isoelectric CAs form acontinuous chain as the electric field would tie them totheir isoelectric zones while diffusion would cause themto broaden just enough to penetrate the neighboringampholytes, thus simultaneously ensuring buffer capacityand conductivity. The result was not just a few movingboundaries, la Tiselius, but a horde of stationaryboundaries, each one standing guard against local pHchanges. It was too bad that this army was barely ahandful of soldiers, hardly able to cover the grounds in thepH 310 interval. Nonetheless, Svensson published in1964 some remarkable color pictures of unique Hbseparations in his 110-mL focusing column stabilized by asucrose density gradient. In these separations, Harryused protein lysates, notably of casein, albumin, Hb, andwhole blood, as background CAs. Peptides rich in Hisprovided the much needed buffering power and con-ductivity in the pH 67 gap [13].

    This is a brief excursus of the many years of labor andintellectual challenge that haunted Harry. But how did hefinally get to the steady state? With a somersault, just like

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    any Italian would have done! He realized early enough inthe game that it would have been extremely difficult tosimulate and solve the transient state, i.e. the kineticbehavior of charged amphoteric molecules during theirtransport by the electric field in an IEF system. So, hewisely chose to solve the equation of the concentrationdistribution of the amphoteric ion in a stationary position,i.e. once this species had reached its pI along the pHgra