8 10
G.Espinosa eta/.
Electrophoresis 1990, 11, 810-812
Georgina Espinosa' Elisabeth WenischZ Herbert Dannet2 Hermann Katinger2 Pier Giorgio Righetti3
Isoelectric focusing in immobilized pH gradients of phosphoglucomutaseand esterasesfrom the spiny lobster
'Faculty of Biology, University of Habana 21nstitute of Applied Microbiology, University of Agriculture and Forestry, Vienna 3Departmentof Biomedical Sciences and Technologies, University of Milano
A method is described for detecting polymorphisms ofcephalothorax and tail homogenates of 25 puerulus staged Panulirus argus in phosphoglucomutase (PGM) and esterases. Isoelectric focusing in immobilized pH gradients was used. In the pH 6.0-8.0 interval for phosphoglucomutase and in the pH 3.5-5.0 and 4.2-4.9 ranges for esterases, both enzymes appeared as polymorphic band patterns. These could be explained by one locus with 2 alleles for phosphoglucomutase and 3 loci with 2,3 and 4 alleles for esterases. Esterases exhibit a more extensive polymorphism in immobilized pH gradients than in polyacrylamide gel electrophoresis.
1 Introduction The spiny lobster (Panulirus argus) is an important natural resource of countries in the Carribean area. When characterizing this species it is important to recognize polymorphic loci. Some populations of this crustacean have been analyzed by conventional electrophoresis [ 1, 21. Isoenzymes are excellent markers of species variability, but - under standard electrophoretic conditions - only one third of the alleles resulting from amino acid substitutions can be detected. Isoelectric focusing (IEF) in immobilized pH gradients (IPG) was developed in order to increase the chance of detecting these silent electromorphs [3]. The present study was undertaken to determine whether esterases and phosphoglucomutases (PGM) show a greater variability with this technique than with standard electrophoretic methods and, subsequently, to obtain a better separation ofthese loci by means ofIPG.
2 Materials and methods 2.1 Animal collection and tissue preparation Twenty five puerulus stage (>4 weeks of age) P. argus were collected in Cuba with the collaboration of the Centre of Marine Research, University of Habana and were stored frozen. For analysis, total tail or total cephalothorax without legs and antennae were homogenized in 1-2 volumes (depending on the tissue) of distilled water. The homogenate was centrifuged for 30 min at 10 OOOg in a centrifuge at +4 OC and the supernatant was frozen and saved for subsequent electrophoretic analyses.
2.2 Equipment and chemicals for electrophoresis All IPG experiments were performed in the LKB 2 1 17 Multiphor I1 horizontal electrophoresis system together with the LKB 2297 Macrodrive 5 power supply and MultitempII thermostat. IPG gel casting was carried out by using the LKB 2 1 17-903 two-dimensional gradient and Immobiline gel kit. Correspondence:Dr. E. Wenisch, Institute of Applied Microbiology, University of Agriculture and Forestry, Peter-Jordan-Strasse 82, A- I I YO Vienna, Austria
Abbreviations: Est, esterase; IEF, isoelectric focusing; IPG, immobilized pH gradient; MTT, 13-(4,5)-dimethylthiazolyn-2,5 diphenyl tetrazolium bromide1 ;PGM, phosphoglucomutase; PMS, phenazine methasulfate; PI, isoelectric point 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
Acrylamide, N,N'-methylenebisacrylamide(Bis), N,N,N',N'tetramethylethylenediamine (TEMED), Repel-Silane, GelBond PAG film, Immobilines and Pharmalytes were purchased from Pharmacia-LKR Biotechnology (Uppsala, Sweden). a- and P-Naphthyl acetate, Fast Blue RR salt, glucose1-phosphate, NADP (nicotinamide-adenine dinucleotide phosphate), I 3-(4,5)-dimethyl-thiazolyn-2,5-diphenyltetrazolium bromidel (MTT) and phenazine methasulfate (PMS) were obtained from Sigma (St. Louis, MO). All other chemicals used were of high purity grade.
2.3 Analytical IEF in IPGs IPG gels (250 x 100 x 0.5 mm) with 5 % T and 2.5 % C were cast. Immobiline pH 6.0-8.0 (PGM) and pH 3.5-5.0 and 4.2-4.9 (esterase) gradients were formed as described in [41. After pouring, the gels were polymerized for 1 h at 50 OC. The gels were then washed 9 times with 250 mL distilled water for 30 min and with 250 mL of 1 O h glycerol for 1 h. The gels were dried with a cold fan (1-2 h) and rehydrated in 30 % v/v glycerol, 2 % w/v Pharmalyte pH 5.0-8.0 for PGM, or in 30 % v/v glycerol, 2 % w/v Ampholine pH 3.5-6 for esterases, for at least 2 h in the reswelling casette (LKB). The use of high levels of sorbitol, or of glycerol, in IPGs was described by Mosher et al. [91 and by Kinzkofer-Pereschet al. [ 101, respectively. The electrode strips werewetted with distilled water. Five pL (PGM) and 15 FL (esterases) ofthe extracts were used and applied onto the gel surface with LKB 2 1 172 15 IEF applicator strips. Sample loading was for 2 h at constant 400 V, followed by constant 2500 V. Focusing was continued for 7 h for the pH 6-8 and 3.5-5 IPG intervals, while twice that time was allowed for focusing in the IPG 4.2-4.9 range. The focusing chamber was flushed with argon when running the pH 6-8 gels.
2.4 Enzyme staining The PGM visualization was performed as recommended in [5 I. Esterase staining was according to [61 with some modifications: 30 min at 37 "C in a plastic tray containing 0.03 % a-naphthyl acetate, 0.015 % P-naphthyl acetate and 0.08 % Fast Blue RR in 0.2 M phosphate buffer, pH 6.5-7.0. Both a- and P-naphthyl acetate were previously dissolved in 2 mL of absolute ethanol. Before staining, the gel was washed for 1-2 min with the staining buffer. Thereafter the gel was fixed with 8 YOacetic acid and dried at room temperature overnight. 0173-0835/90/0909 0810 $3.50+.25/0
Phosphoglucoinutnse arid eyterases from the spiny lobster
Electrophoresis 1990,l I , 810-81 2
8 11
3 Results and discussion The PGM assay revealed four zones, A to D, beginning from the most anodal zone of enzymatic activity (Fig. 1). The isoelectric point (PI)of the most anodal zone was 6.32. This and the most cathodal one (pl7.30 and 7.37) did not change among the different specimen. The two zones in the middle (B and C) exhibited polymorphism. Two alleles were identified. One heterozygous phenotype could be observed in Fig. 1. The p l of alleles of zone B were 6.59 for the most frequent allele and 6.62 for the other one. The upper bands of zone C (pl 6.82 and 6.90) showed the same polymorphic behavior as zone B. The zone also showed another monomorphic band with p16.96. We found a frequency of 0.06 for the rare allele which fits the Hardy-Weinberg equilibrium. The average heterozygosity for this enzyme was 0.13, which is similar to the previously found value for this species using starch gel electrophoresis [ 1, 21. PGM has been analyzed using IPGs 171 in forensic science, where the 4 common alleles of this enzyme from human erythrocytes were detected. We cannot estimate whether the degree of separation was better or not compared to starch gels electrophoresis [ 1, 21.
Figure 1. Photograph of a portion of a polyacrylamide gel in the IPG pH 6.0-8.0 interval, stained to detect PGM activity of tail extracts from puerulus stage Panulirus argus. Zone B and the upper bands ofzone c are polymorphic. Phenotype No. 1 (from left) was found to be heterozygous. The anode is at the bottom.
In Fig. 2 the esterase electropherograms of cephalothorax of several specimens are shown. We could observe two polymorphic esterase zones. The third zone is shown in Fig. 3. These zones were named: Est-A, Est-B and Est-C, in the order of increasing PIS. Esterases showed a plof 4.00-4.20 for EstA, 4.32-4.42 for Est-B and 4.55-4.61 for Est-C. The variations in the three zones appear to occur largely independently of each other in the population and it is therefore most likely that they were determined by three different genetic systems. The most anodal zone, Est-A, is the widest one and showed a complex pattern, with varying numbers of bands. We found 4 simple phenotypes with 8 bands and with different pls for these major bands. They were named a, b, c and d. Phenotype a is composed of the lowest p 1 bands. We identify each different group Of ‘Ones with One Four ‘Ombinations o f t h e six possible Ones were f(X.lnd. In Fig. 2 we observe that the most frequent allele was C and the most frequent combination was b with c (phenotype b/c). This allele was also combined with a and d. We could observe 8 out of 10
Figure 2. Photograph of a portion of a polyacrylamide gel in the I P G pH 3.5-5.0 interval, stained to detect esterase activity of cephalothorax’extractsfrom puerulus stage Panulirus argus. The phenotypes from Est-A are in the lower zone and Est-B in the upper part of the photograph. The most frequent phenotypes are: Est-A, c and b/c; Est-B, b and a/b. The anode is at the bottom.
8 12
G. Espinosa e f a1
Electrophoresis 1990, 11. 8 10-8 I2
Table 1. Observed and expected phenotypes for Hardy-Weinberg equilibrium of esterase loci and allele frequency in a Cuban population of puerulus of Panulirus argus
Esterase
ala
bib
Phenotypes clc aib
alc
bic
0
3
Allele frequency a b c
~~
Obs
5
8
0
1
Exp.
3.8
8.1
0.1
11.1
1.1
1.7
4
-
10
-
-
0
B Obs
10
C EXP
9.3
3.4
0.38
0.56
0.37
0.63
0.06
11.2
these esterase systems with ours, we calculated the average heterozygosity for esterases by taking the allele frequencies published by Menzies and Kerrigan 11 and those observed by us. We obtained values of 0.25 for vertical polyacrylamide gel electrophoresis and 0.56 for our system. This means that IEF in IPGs can reveal a higher degree of genetic variability than other electrophoretic methods, in agreement with IS].
4 Conclusions _ -
Figure 3. Photograph of a portion of a polyacrylamide gel in the IPG pH 4.2-4.9 interval, stained to detect esterase activity of cephalothorax extracts from puerulus stage Panulirus argus, showing the three phenotypes of Est-C. The anode is at the bottom.
phenotypes, assuming the simplest genetic system to have 4 alleles. Having only analyzed 20 samples, we did not calculate allelic frequency. Nevertheless, the observed phenotypic distributions follow the Hardy-Weinberg proportions. Four phenotypes of Est-B occur in the investigated populations: two with a single band and two with a double band. One double-banded phenotype contains an allele which is different from the single-banded ones (Fig. 2). Based on this behavior we can assume three different alleles (a, b, and c) for thislocus. This zone can be easily detected with an lmmobiline pH 3.5-5.0 gel, but also with an Immobiline pH 4.2-4.9 gel. The frequencies of these alleles are shown in Table 1. All genotypic proportions agree well with the Hardy-Weinberg expectations. The zone called Est-C showed three different phenotypes, two with a single band and one with a double band. It is reasonable to assume that the variation in this region is due to a single gene with two codominant alleles (Fig. 3). This zone can be detected clearly with an lmrnobiline pH 4.2-4.9 gel. The frequency of these alleles is shown in Table 1. All genotypic proportions comply well with the HardyWeinberg expectations. Three esterase systems were described for P. argus using vertical polyacrylamide gels 11,2]. With IPG we most probably found the same systems as these authors. In order to compare
The polymorphism of PGM and of esterases ofP. argus can be clearly detected by using IPGs in the pH 6.0-8.0 interval for PGM and 3.5-5.0 or 4.2-4.9 for esterases. For this last enzyme, this technique can turn up a substantially greater variability than conventional polyacrylamide gel electrophoresis.
G. Espinosa was the recipient of a researchfellowshipfrom the International Atomic Energy Agency. PGR is supported by a grant from Progetto Finalizzato Chimica Fine II (CNR, Roma). Received February 19, 1990
5 References II 1 Menzies, R. A. and Kerrigan, J. M., Proc. Gu& Carib. Fish. Inst. 1979,31, 164-178. [21 Menzies, R. A,, Proc. Gulf. Carib. Fish. Inst. 1981,33,230-243. 131 Righetti, P. G., in: Creighton, T. E., (Ed.),Protein Struciure:A Practicai Approach IRL Press, Oxford, 1989. pp. 23-63. 141 Righetti, P. G. and Gianazza, E., Methods Biochem. Anal. 1987,32. 215-278. [ 5 1 Sutton,J. G. and Westwood, S. A.,Electrophoresis 1984,5,252-253. [61 Shaw, C. R. and Prasad, R., Biochem. Genet. 1970,4,297-320. [71 Westwood, S. A., Sutton, J . G. and Gill, P., in: Neuhoff, V. (Ed.), Electrophoresis '84, VCH, Weinheim 1984, pp. 98-101. [8l Righetti, P. G., Isoelectric Focusing: Theory, Methodology and A p plicafions. Elsevier, Amsterdam, 1983, pp. 225-235. [91 Mosher, R. A., Bier, M. and Righetti, P. G., Electrophoresis 1986. 7 , 59-66. I101 Kinzkofer Peresch, A., Patestos, N. P., Fauth, M., KogeLF., Zok,R. and Radola, B. J., Electrophoresis 1988, 9,497-5 11.
G.Espinosa eta/.
Electrophoresis 1990, 11, 810-812
Georgina Espinosa' Elisabeth WenischZ Herbert Dannet2 Hermann Katinger2 Pier Giorgio Righetti3
Isoelectric focusing in immobilized pH gradients of phosphoglucomutaseand esterasesfrom the spiny lobster
'Faculty of Biology, University of Habana 21nstitute of Applied Microbiology, University of Agriculture and Forestry, Vienna 3Departmentof Biomedical Sciences and Technologies, University of Milano
A method is described for detecting polymorphisms ofcephalothorax and tail homogenates of 25 puerulus staged Panulirus argus in phosphoglucomutase (PGM) and esterases. Isoelectric focusing in immobilized pH gradients was used. In the pH 6.0-8.0 interval for phosphoglucomutase and in the pH 3.5-5.0 and 4.2-4.9 ranges for esterases, both enzymes appeared as polymorphic band patterns. These could be explained by one locus with 2 alleles for phosphoglucomutase and 3 loci with 2,3 and 4 alleles for esterases. Esterases exhibit a more extensive polymorphism in immobilized pH gradients than in polyacrylamide gel electrophoresis.
1 Introduction The spiny lobster (Panulirus argus) is an important natural resource of countries in the Carribean area. When characterizing this species it is important to recognize polymorphic loci. Some populations of this crustacean have been analyzed by conventional electrophoresis [ 1, 21. Isoenzymes are excellent markers of species variability, but - under standard electrophoretic conditions - only one third of the alleles resulting from amino acid substitutions can be detected. Isoelectric focusing (IEF) in immobilized pH gradients (IPG) was developed in order to increase the chance of detecting these silent electromorphs [3]. The present study was undertaken to determine whether esterases and phosphoglucomutases (PGM) show a greater variability with this technique than with standard electrophoretic methods and, subsequently, to obtain a better separation ofthese loci by means ofIPG.
2 Materials and methods 2.1 Animal collection and tissue preparation Twenty five puerulus stage (>4 weeks of age) P. argus were collected in Cuba with the collaboration of the Centre of Marine Research, University of Habana and were stored frozen. For analysis, total tail or total cephalothorax without legs and antennae were homogenized in 1-2 volumes (depending on the tissue) of distilled water. The homogenate was centrifuged for 30 min at 10 OOOg in a centrifuge at +4 OC and the supernatant was frozen and saved for subsequent electrophoretic analyses.
2.2 Equipment and chemicals for electrophoresis All IPG experiments were performed in the LKB 2 1 17 Multiphor I1 horizontal electrophoresis system together with the LKB 2297 Macrodrive 5 power supply and MultitempII thermostat. IPG gel casting was carried out by using the LKB 2 1 17-903 two-dimensional gradient and Immobiline gel kit. Correspondence:Dr. E. Wenisch, Institute of Applied Microbiology, University of Agriculture and Forestry, Peter-Jordan-Strasse 82, A- I I YO Vienna, Austria
Abbreviations: Est, esterase; IEF, isoelectric focusing; IPG, immobilized pH gradient; MTT, 13-(4,5)-dimethylthiazolyn-2,5 diphenyl tetrazolium bromide1 ;PGM, phosphoglucomutase; PMS, phenazine methasulfate; PI, isoelectric point 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
Acrylamide, N,N'-methylenebisacrylamide(Bis), N,N,N',N'tetramethylethylenediamine (TEMED), Repel-Silane, GelBond PAG film, Immobilines and Pharmalytes were purchased from Pharmacia-LKR Biotechnology (Uppsala, Sweden). a- and P-Naphthyl acetate, Fast Blue RR salt, glucose1-phosphate, NADP (nicotinamide-adenine dinucleotide phosphate), I 3-(4,5)-dimethyl-thiazolyn-2,5-diphenyltetrazolium bromidel (MTT) and phenazine methasulfate (PMS) were obtained from Sigma (St. Louis, MO). All other chemicals used were of high purity grade.
2.3 Analytical IEF in IPGs IPG gels (250 x 100 x 0.5 mm) with 5 % T and 2.5 % C were cast. Immobiline pH 6.0-8.0 (PGM) and pH 3.5-5.0 and 4.2-4.9 (esterase) gradients were formed as described in [41. After pouring, the gels were polymerized for 1 h at 50 OC. The gels were then washed 9 times with 250 mL distilled water for 30 min and with 250 mL of 1 O h glycerol for 1 h. The gels were dried with a cold fan (1-2 h) and rehydrated in 30 % v/v glycerol, 2 % w/v Pharmalyte pH 5.0-8.0 for PGM, or in 30 % v/v glycerol, 2 % w/v Ampholine pH 3.5-6 for esterases, for at least 2 h in the reswelling casette (LKB). The use of high levels of sorbitol, or of glycerol, in IPGs was described by Mosher et al. [91 and by Kinzkofer-Pereschet al. [ 101, respectively. The electrode strips werewetted with distilled water. Five pL (PGM) and 15 FL (esterases) ofthe extracts were used and applied onto the gel surface with LKB 2 1 172 15 IEF applicator strips. Sample loading was for 2 h at constant 400 V, followed by constant 2500 V. Focusing was continued for 7 h for the pH 6-8 and 3.5-5 IPG intervals, while twice that time was allowed for focusing in the IPG 4.2-4.9 range. The focusing chamber was flushed with argon when running the pH 6-8 gels.
2.4 Enzyme staining The PGM visualization was performed as recommended in [5 I. Esterase staining was according to [61 with some modifications: 30 min at 37 "C in a plastic tray containing 0.03 % a-naphthyl acetate, 0.015 % P-naphthyl acetate and 0.08 % Fast Blue RR in 0.2 M phosphate buffer, pH 6.5-7.0. Both a- and P-naphthyl acetate were previously dissolved in 2 mL of absolute ethanol. Before staining, the gel was washed for 1-2 min with the staining buffer. Thereafter the gel was fixed with 8 YOacetic acid and dried at room temperature overnight. 0173-0835/90/0909 0810 $3.50+.25/0
Phosphoglucoinutnse arid eyterases from the spiny lobster
Electrophoresis 1990,l I , 810-81 2
8 11
3 Results and discussion The PGM assay revealed four zones, A to D, beginning from the most anodal zone of enzymatic activity (Fig. 1). The isoelectric point (PI)of the most anodal zone was 6.32. This and the most cathodal one (pl7.30 and 7.37) did not change among the different specimen. The two zones in the middle (B and C) exhibited polymorphism. Two alleles were identified. One heterozygous phenotype could be observed in Fig. 1. The p l of alleles of zone B were 6.59 for the most frequent allele and 6.62 for the other one. The upper bands of zone C (pl 6.82 and 6.90) showed the same polymorphic behavior as zone B. The zone also showed another monomorphic band with p16.96. We found a frequency of 0.06 for the rare allele which fits the Hardy-Weinberg equilibrium. The average heterozygosity for this enzyme was 0.13, which is similar to the previously found value for this species using starch gel electrophoresis [ 1, 21. PGM has been analyzed using IPGs 171 in forensic science, where the 4 common alleles of this enzyme from human erythrocytes were detected. We cannot estimate whether the degree of separation was better or not compared to starch gels electrophoresis [ 1, 21.
Figure 1. Photograph of a portion of a polyacrylamide gel in the IPG pH 6.0-8.0 interval, stained to detect PGM activity of tail extracts from puerulus stage Panulirus argus. Zone B and the upper bands ofzone c are polymorphic. Phenotype No. 1 (from left) was found to be heterozygous. The anode is at the bottom.
In Fig. 2 the esterase electropherograms of cephalothorax of several specimens are shown. We could observe two polymorphic esterase zones. The third zone is shown in Fig. 3. These zones were named: Est-A, Est-B and Est-C, in the order of increasing PIS. Esterases showed a plof 4.00-4.20 for EstA, 4.32-4.42 for Est-B and 4.55-4.61 for Est-C. The variations in the three zones appear to occur largely independently of each other in the population and it is therefore most likely that they were determined by three different genetic systems. The most anodal zone, Est-A, is the widest one and showed a complex pattern, with varying numbers of bands. We found 4 simple phenotypes with 8 bands and with different pls for these major bands. They were named a, b, c and d. Phenotype a is composed of the lowest p 1 bands. We identify each different group Of ‘Ones with One Four ‘Ombinations o f t h e six possible Ones were f(X.lnd. In Fig. 2 we observe that the most frequent allele was C and the most frequent combination was b with c (phenotype b/c). This allele was also combined with a and d. We could observe 8 out of 10
Figure 2. Photograph of a portion of a polyacrylamide gel in the I P G pH 3.5-5.0 interval, stained to detect esterase activity of cephalothorax’extractsfrom puerulus stage Panulirus argus. The phenotypes from Est-A are in the lower zone and Est-B in the upper part of the photograph. The most frequent phenotypes are: Est-A, c and b/c; Est-B, b and a/b. The anode is at the bottom.
8 12
G. Espinosa e f a1
Electrophoresis 1990, 11. 8 10-8 I2
Table 1. Observed and expected phenotypes for Hardy-Weinberg equilibrium of esterase loci and allele frequency in a Cuban population of puerulus of Panulirus argus
Esterase
ala
bib
Phenotypes clc aib
alc
bic
0
3
Allele frequency a b c
~~
Obs
5
8
0
1
Exp.
3.8
8.1
0.1
11.1
1.1
1.7
4
-
10
-
-
0
B Obs
10
C EXP
9.3
3.4
0.38
0.56
0.37
0.63
0.06
11.2
these esterase systems with ours, we calculated the average heterozygosity for esterases by taking the allele frequencies published by Menzies and Kerrigan 11 and those observed by us. We obtained values of 0.25 for vertical polyacrylamide gel electrophoresis and 0.56 for our system. This means that IEF in IPGs can reveal a higher degree of genetic variability than other electrophoretic methods, in agreement with IS].
4 Conclusions _ -
Figure 3. Photograph of a portion of a polyacrylamide gel in the IPG pH 4.2-4.9 interval, stained to detect esterase activity of cephalothorax extracts from puerulus stage Panulirus argus, showing the three phenotypes of Est-C. The anode is at the bottom.
phenotypes, assuming the simplest genetic system to have 4 alleles. Having only analyzed 20 samples, we did not calculate allelic frequency. Nevertheless, the observed phenotypic distributions follow the Hardy-Weinberg proportions. Four phenotypes of Est-B occur in the investigated populations: two with a single band and two with a double band. One double-banded phenotype contains an allele which is different from the single-banded ones (Fig. 2). Based on this behavior we can assume three different alleles (a, b, and c) for thislocus. This zone can be easily detected with an lmmobiline pH 3.5-5.0 gel, but also with an Immobiline pH 4.2-4.9 gel. The frequencies of these alleles are shown in Table 1. All genotypic proportions agree well with the Hardy-Weinberg expectations. The zone called Est-C showed three different phenotypes, two with a single band and one with a double band. It is reasonable to assume that the variation in this region is due to a single gene with two codominant alleles (Fig. 3). This zone can be detected clearly with an lmrnobiline pH 4.2-4.9 gel. The frequency of these alleles is shown in Table 1. All genotypic proportions comply well with the HardyWeinberg expectations. Three esterase systems were described for P. argus using vertical polyacrylamide gels 11,2]. With IPG we most probably found the same systems as these authors. In order to compare
The polymorphism of PGM and of esterases ofP. argus can be clearly detected by using IPGs in the pH 6.0-8.0 interval for PGM and 3.5-5.0 or 4.2-4.9 for esterases. For this last enzyme, this technique can turn up a substantially greater variability than conventional polyacrylamide gel electrophoresis.
G. Espinosa was the recipient of a researchfellowshipfrom the International Atomic Energy Agency. PGR is supported by a grant from Progetto Finalizzato Chimica Fine II (CNR, Roma). Received February 19, 1990
5 References II 1 Menzies, R. A. and Kerrigan, J. M., Proc. Gu& Carib. Fish. Inst. 1979,31, 164-178. [21 Menzies, R. A,, Proc. Gulf. Carib. Fish. Inst. 1981,33,230-243. 131 Righetti, P. G., in: Creighton, T. E., (Ed.),Protein Struciure:A Practicai Approach IRL Press, Oxford, 1989. pp. 23-63. 141 Righetti, P. G. and Gianazza, E., Methods Biochem. Anal. 1987,32. 215-278. [ 5 1 Sutton,J. G. and Westwood, S. A.,Electrophoresis 1984,5,252-253. [61 Shaw, C. R. and Prasad, R., Biochem. Genet. 1970,4,297-320. [71 Westwood, S. A., Sutton, J . G. and Gill, P., in: Neuhoff, V. (Ed.), Electrophoresis '84, VCH, Weinheim 1984, pp. 98-101. [8l Righetti, P. G., Isoelectric Focusing: Theory, Methodology and A p plicafions. Elsevier, Amsterdam, 1983, pp. 225-235. [91 Mosher, R. A., Bier, M. and Righetti, P. G., Electrophoresis 1986. 7 , 59-66. I101 Kinzkofer Peresch, A., Patestos, N. P., Fauth, M., KogeLF., Zok,R. and Radola, B. J., Electrophoresis 1988, 9,497-5 11.
