214

these can be used to identify the components in the protein mixture being investigated.

Various specific staining techniques are available, but of these the potentially most powerful are those based on immunological cross-reactivity. This approach has been revolutionized by blotting tech- ‘niques for transferring proteins from gels onto substrates such as nitrocellulose. In this state the proteins are readily accessible to probes such as specific polyclonal and monoclonal antisera. The immunocomplexes formed can be visualized using a second antibody or protein A labelled with [‘““I], horseradish peroxidase or fluorescein isothiocyanate.

It is possible to excise and recover selected protein spots from 2D gels. Proteins isolated in this way can be readily characterized by enzyme analysis, amino acid analysis, peptide mapping, and protein sequencing, providing that suitable microanalytical techniques are available. In addition, monoclonal antibodies can be raised against the recovered proteins.

Conclusion Recent developments in 2D-PAGE make it possible

to analyse complex protein mixtures. The technique is therefore a potent research tool in many areas of biological and biomedical research, but problems remain to be resolved and procedures standardized before 2D-PAGE can be considered to be a routine procedure. Nevertheless, the high resolution capacity

trends in analytical chemistry, uol. 2, no. 9’ 1983

of 2D-PAGE for proteins and recent advances in genetic engineering techniques for studies of genome organization now provide methods for detailed analysis of both genotypic and phenotypic expression.

Acknowledgements We gratefully acknowledge financial support from

the Muscular Dystrophy Group of Great Britain and the Medical Research Council.

References 1 O’Farrell, P. H. (1975)j. Biol. Chem. 250, 4017 2 Dunn, M. J. and Burghes, A. H. M. (1983) Electrophoresis 4,97 3 Dunn, M. J. and Burghes, A. H. M. (1983) Electrophoresis 4,173 4 O’Farrell, P., Goodman, M. M. and O’Farrell, P. H. (1977) Cell

12, 1133

5 Burghes, A. H. M., Dunn, M. J. and Dubowitz, V. (1982) Electrophoresis 3, 354

6 Cuono, C. B. and Chapo, G. A. (1982) Electrophoresis 3, 65 7 Anderson, N. L. and Anderson, N. G. (1978) Anal. Biochem. 85,

331 8 Anderson, N. L. and Anderson, N. G. (1978) Anal. Biochem. 85,

341 9 Anderson, N. L., Taylor, J., Scandora, A. E., Coulter, B. P. and

Anderson, N. G. (1981) Clin. Gem. (Winston Salem. NC) 27, 1807 10 McConkey, E. H. (1976) Anal. Biochem. 96, 39

Michael J. Dunn and Arthur H. M. Burghes are members of th research staff of the Jerry Lewis Muscle Research Centre at the Royal Postgraduate Medical School, Duane Road, London WI2 OHS, UK. Dr Dunn is President of the British Electrophoresis Society.

Atmlications of electrofocusina &&focusing is an electrophoretic technique in which y protein or peptide migrates through a pH gradient under the influence of an electric current until it reaches the pH where its net charge is zero, this is the isoelectric point, pl. Inherent in electrofocusing is a concentrating effect which coun- teracts diffusion and provides much greater resolution than most other electrophoretic techniques. It has been used to great advantage in the isolation and identification of genetic

variants of many proteins. Roger Bishop Bromma, Sweden

Since carrier ampholytes and equipment for elec- trofocusing first became commercially available in 1966, well over 5 000 articles1 and several application reviews2*3 on the technique have been published. To avoid repetition, in this article I would like to describe some of the most recent and novel applications of electrofocusing.

Electrofocusing in biochemistry, for its own sake Estimates of the smallest difference in isoelectric

point for two proteins which can just be separated are about 0.01 pH units for carrier-ampholyte based systems (e.g. ‘LKB Ampholine’ carrier ampholytes) and 0.001 pH units for immobilized pH gradients 0165~~36/83/$01.00

provided by the recently-introduced ‘LKB Immobiline’ System. Thus, electrofocusing has been used for the analysis of all kinds of proteins, from acetaldehyde dehydrogenase to zein.

Electrofocusing is also widely used in the preparative purification of proteins, because of the combination of high resolution and the concentrating effect, as well as its simplicity of use. Unlike such techniques as gel permeation and ion exchange chromatography, the resolution in electrofocusing does not fall greatly as the amount of sample applied is increased. For example, in our laboratories, 7 ml of serum has been fractionated on a 5 mm-thick preparative Immobiline gel slab with a pH gradient of pH 4-6, whilst retaining the resolution typical of a 0.5 mm-thick analytical polyacrylamide gel (Ek, K., Bjellqvist, B. and Righetti, P. G., unpublished observations). Fig. 1 shows another example of

0 1983 Elsevicr Science Publishers B.V.

trcna? in dytical hnzistty, vol. 2, no. 9,198.J 215

Fig. 1. Preparative pw$iation of commtrcially-availablc horse heart myoglobin by ckectrofocusing in an immobilizedpH6.8-7.8Igradiml, using the LKB Immobilinc Z&tern. Gel contains 5%Tand 3%C, it is 5 mm thick and has a bufftig ca#acit_v of 6 mEq 1-l per pH unit. Sample size was 200 mg, a&lied in th slot near the anoa?. Running conditiot~ were: maxima of 2 5tM V, 5 mA and 5 W, at IO”C ovcmight. Staining was with Coomassie Brilliant Blue R-250. Courtesy of Dr Kristina Ek, LKB-Produkter AB.

preparative electrofocusing. Thus, by replacing two or three poorly-resolving and/or time-consuming steps in a protein purification scheme, a single electrofocusing run can provide much purer and ‘fresher’ proteins.

Screening, diagnosis and drug testing The high resolving power ofelectrofocusing has been

successfully applied to the differentiation of genetic variants of many clinically interesting proteins. Examples range from haemoglobin, which gives a single band (i.e. HbA) on electrofocusing, to polymorphic proteins, such as al-antitrypsin2, which gives eight bands. Studies of protein polymorphism for screening purposes are likely to increase because of the known relationship between certain phenotypes and susceptibility to disease; for example, the link between phosphoglucomutase ( PGMx) and tuberculosis4.

Assays of a normal haemoglobin are widely used in the management of diabetic patients5. HbAlc is formed in the blood by a non-enzymatic reaction between normal adult haemoglobin (HbA) and circulating glucose, and is usually found in elevated concentrations in diabetics. The concentration of HbArc is correlated with the mean blood glucose level over two or three weeks before treatment, and is thus a better indicator of the degree of diabetic control than spot measurements of blood glucose. Although HbA and HbAlc differ very little in isoelectric point, the use of a very narrow pH gradient for electrofocusing gives sufficient resolution for subsequent quantitation.

Electrofocusing has been used in investigations to optimize the structure of potential antisickle cell anaemia agents’; the binding of 21 such agents to the accessible thiol groups of haemoglobin was assessed by comparing the electrofocusing band patterns of modified and native haemoglobin.

Who dunnit? Forensic science and paternity testing are other

practical examples where genetic population studies of proteins are used. Although blood group testing is perhaps more readily done by serology, electrofocusing methods have now been developed for a large number of genetic variants of other proteins.

While whole blood is a common sample source in forensic science, it is not always available. The UK Home Oflice Central Research Establishment at Aldermaston has developed a technique which uses the sheath cells around the root of a hair; these are often carried with the hair when it is torn from the scalp in acts ofviolence. A single hair is inserted into the gel slab used for electrofocusing. Initially, the technique was developed for the protein phosphoglucomutase and a single hair was used for every run. Nowadays, the single hair can be cut into sections, each of which is used in the analysis of a different enzyme system’.

The analysis of haemoglobin from autopsy material may be useful in determining the cause ofdeath, since it is possible to differentiate and distinguish derivatives such as CO-Hb and Sulph-Hb, resulting from the inhalation of carbon monoxide or hydrogen sulphide*.

Food for thought The species-specific band patterns produced by

electrofocusing proteins present in skeletal muscle or storage proteins has provided the analytical chemist with an excellent method for distinguishing and identiftring fish and animal meats, as well as cereals, potatoes, fruit and vegetable9. Of great interest to the consumer, as well as to the Weights and Measures Inspector, is the ability to detect very low concentra- tions of adulterants or ‘extenders’ in a large variety of foodstuffs: for example, cows’ milk in sheep or goats’ milk; rice or buckwheat flour in wheat flour; kangaroo, horse, pig or buffalo meat in beet and soya or other non-meat proteins in beef. At present, however, such studies can only be done on raw fresh or frozen meats; the one exception being crab meat, the genera ofwhich can still be identified even after cooking and removal of the meat from the skeleton.

The ripening of Camembert cheese is a complex process, involving the proteolysis of casein by proteins from a diversity of sources: native milk proteinases (essentially plasmin); milk clotting enzymes present in the rennet (bovine pepsin and chymosin); and proteinases of the microflora (where the aspartyl- and metallo-proteinases of Penidium ca.w’colum play the leading roles in proteolysis). In order to characterize the action of the different proteinases, Trieu-Cuot and co-workers at the Institut National de la Recherche Agronomique, Jouy-en-Josas, first studied in vitro the separate effects on isolated caseins of proteinases from the different sources, using electrofocusing and two-dimensional electrophoresis. The breakdow? patterns and kinetics could be easily followed, the casein components p1 values were determined and the degradation products identified. Using the same

216 trends in analytical chemistry, WI. 2, no. 9; 1983

Unidentified products

PA’mp2a + PAlmp2b + PAlap

.I~A’/A’ -

y,A’IA= \ 7 ; . . - am- w

..m..---me---

Y--=--_.-. --I

=..m.--IE”I --

- rcec- -- w- -- -

flA’mp3 + r,A’/A’

pAl mp4 + y,A’ + @A ,*mp3)

PAImp + PA’ap3

(PA2mp4 + y,A*)

4P --I 1 1 A IS CllS Cl1 S CIIS Cl1 s ~11s c/I~~c~/s,~cj 3 4 5 6 7 8 \‘-- (PA’mp5+PAZw3)

L- %I + %#P3

Fig. 2. Electrofocusing of thepH 4.6~insolublefraction of Camembert cheeses: 1, whole casein; 2, curd after 6 h of draining; 3-10, Camembert cheeses after 1,3,7, 10,14,21,28 and 35 d of ripening, respectively. Proteolysis was studied separately in the surface (S) and centre (C) of the cheese. The casein breakdown products

indicated on the right are identz$iid from earlier work. Some minor degradation products from PA’-casein are not mentioned or are indicated in parentheses. Reproduced with permission of authors and publisher fromJ. Dairy Res. (1982) 49, 501.

techniques, the action of these enzymes during the ripening of Camembert cheese’ could then be followed (Fig. 2). Rennet acted promptly on asl-casein, with the (~~11 breakdown product appearing within 6 h of draining the curd. Proteolysis by P. caseicolum occurred after 7 days on the surface of the cheese, but was delayed until 28-35 days in the centre; similarly, breakdown products produced by milk plasmin appeared only after 2 l-35 days on the surface and after 35 days in the centre, indicating the influence of the changing pH within the maturing cheese.

Molecular biology and epidemiology of viruses The foot-and-mouth disease virus (FMDV) is ideal

for study by electrofocusing for several reasons: the virus-induced polypeptides can be focused into sharp, usually single bands; since FMDV produces only a single species of polycistronic messenger RNA, its genes are conveniently expressed in equimolar amounts; the number of mature polypeptides is small enough to produce an electrofocusing pattern that can readily be compared in one dimension with that of other strains; finally, the electrofocusing pattern is not complicated by glycosylated derivatives of the polypeptides.

King and co-workers at the Animal Virus Research Institute, Pirbright, Woking, UK have used mutants of FMDV with polypeptides having altered isoelectric points to solve several interesting questions in pure and applied virology”.

Physical mapping of mutations Electrofocusing allows the four FMDV coat

proteins, VPl, VP2, VP3 and VP4, and the major non-structural polypeptides, P34 and P56a, to be resolved. Starting with, for instance, temperature- sensitive (ts) mutants, and examining the altered polypeptides isolated from spontaneous reversions to

the wild type, it was possible to show that ts mutations arise most frequently in the coat protein genes, less frequently in the P56a (RNA polymerase) gene, and rarely in the P34 gene. On a similar theme, guanidine-resistance mutations appear to be exclu- sively confined to the P34 gene.

Precursor-product relationships Mutations affecting mature FMDV polypeptides are

always carried by their precursors.

Genetic recombination in FMDV Mutations producing viral polypeptides with altered

isoelectric points may be used as genetic markers for studying recombination between mutants of the same strain or the same subtype.

Strain identification As applied to the foot-and-mouth disease virus,

strain identification includes the important business of tracking down the source ofthe virus responsible for the outbreak of the disease. One such outbreak started in Brittany and spread, apparently on the wind, to Normandy, Jersey and the Isle of Wight in March 1981. Fig. 3 shows viral polypeptides examined by electrofocusing from virus isolated from these out- breaks, and compared with other isolates of the same serotype. The patterns indicate that the 1981 isolates are very similar to each other; the only differences were in a changed P56a band ofone Isle ofWight isolate, and an extra VP2 band in the Jersey isolate, indicating that the latter may have been a mixture of viruses. Of the other strains examined, the only patterns identical to the 1981 isolates were given by the Lausanne 1965 virus, which is used for vaccine production, and the France 1971 isolate. Since foot-and-mouth disease is not endemic in France, it is unlikely that France 197 1 was the cause of the March 1981 outbreak. On the

trmdr in analytical &n&y, ml. 2, no. 9,198.Y 217

Fig. 3. hhtzhfiation of the virus strain responsible for the outbreaks of foot-and-mouth disease in France and the UK in Mamh 1981. Isolates w4r4 passaged through baby hamster k% (BHK 21) cells labelled witA ,a5S-methionine, cytoplasmit 4xtmts peNred, and viral jn$vpepides precipitated with #&jii antiserum. The washed immunopreti@ztes were incubated in a solution containing urea, mertaptocthanol and NP-40 befwG us4 in elettrofocusing. Thtphotogra~h is a tombositejom the two 4xp4rhnt.s ncedcd to separate tht very basic and the very acidic viraljolyppttidcs: on the right, the inncbation mixture was mixed with wta#toethylammonium thlori& and run in a pH 3.5-10 gel containing 2% NP-40 by non-equilibrium elcctrof~ towark the tathoa!e; forfocuFing of VP2, VP3 and VP4, towardr the anode, the viralpolypeptides were denatured by heating with SDS and run with o@osite @la@. Reprinted by permission from Nature, Vol. 293, No. 5832, p. 479. Copyright ‘@ 1981 Macmillan Journals Limited.

other hand, since passage of FMDV in animals quickly produces detectable changes in polypeptide patterns, it is unlikely that the Lausanne 1965 strain had survived unaltered in the field for 16 years.

Possible conclusions are that the virus escaped from a laboratory, that a vaccine was contaminated with the virus, or that a vaccine itself was incompletely inactivated. In principle, if any of these conclusions is true, it should be possible to identifjr the source more precisely, since stocks of the same strain maintained independently in different laboratories have been found in some cases to give slightly different electrofocusing patterns.

Conclusions There are few areas of pure or applied biochemistry

where electrofocusing has not been used. The number of applications, as well as their novelty, are likely to increase and, with the improved resolution promised by the Immobiline system, so will the ability to distinguish a much larger number ofgenetic variants of proteins.

References Attu Ampholinae, Electrofocusing Reference List 1960-74 (with supplements 1975-77, 1978, 1979, 1980-81) LKB-Pmdukter AB, Box 305, S-161 26 Bromma, Sweden Allen, R. C. (1978)J. Chromatogr. 146, 1 Righetti, P. G. (1981) Elettiophoresir 2, 65 Papiha, S. S., Aganval, S. S. and White, J. (1983)J. Med. &net. 20, 220 Basset, P., Braconnier, F. and Rosa, J. (1982) J. Chromatogr. 227, 267 Garel, M. C., Beuzard, Y., Thillet, J., Domenget, C., Martin, J., Galacteros, F. and Rosa, J. (1982) Eur. J. B&hem. 123, 5 13 Lawton, M. E. and Sutton, J. G. (1982) J. Forensic Sci. Sot. 22, 203 Bonte, W., Jursch, R. and Straube, J. (198 1) Forensic Sti. Znt. 17, 45

9 TrieuCuot, P. and Gripon, J.-C. (1982) J. Dairy Res. 49, 501 10 King, A. M. Q., McCahon, D., Newman, J. W. I., Crowther,

J. R. and Carpenter, W. C. Cur*. Top. Microbial. (in press)

Roger Bishop received his B.Sc. in biochemistryfrom Shtffild Univcrsi& and his Ph.D. from the Uniwrsity of Lea?. Since 1976 hc has been em#oyed by LKB-Prod&v AB, Bromma, Sweah, where h is Manager of the S&ntijii Support Department.