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Electrophoretic titration curve in 6 M urea of the bovine eye lens protein α-crystallin
Journal of Chromatography,
41 I (1987) 507-509
Elsevier Science Publishers B.V., Amsterdam ~ Printed in The Netherlands CHROM. 20 013
Note
Electrophoretic tein a-crystallin
titration
curve
in 6 A4 urea of the bovine
eye lens pro-
PIET J. M. VAN DEN OETELAAR* and HERMAN J. HOENDERS Department of Biochemistry, University of Ndjmegen, Kapittelweg 46,652s
EP Nij,egen
(The Netherlands)
(Received August 25th, 1987)
Recently we reported on the separation of the subunits of the bovine eye lens protein a-crystallin by means of fast protein liquid ehromatography-chromatofocusing in concentrated ureal. We noted that the pH at which the CYAN and aAz subunits eluted from the column differed by 0.41 pH unit from their pZ as determined by isoelectric focusing in concentrated urea, whereas there was no difference for the aB1 chain and only a difference of -0.09 pH unit for the ORBS subunit. Similar observations were made by Bloemendal and Groenewoud2 using a soft-gel PBE-94 column. We suggested that the difference in behaviour of the aA and aB chains is caused by a difference in the net charge of the subunits near their isoelectric point. The eleetrophoretic titration curve of a-crystallin is now presented in order to substantiate this assumption. Electrophoretic titration curves were introduced by Mich13 and have since proved useful for the determination of the optimal pH for other separation techniques such as chromatofocusing, ion-exchange chromatography and electrophoresis and for the detection of isozymes. The first step of the technique consists of the creation of a pH gradient in a horizontal slab gel using ampholytes. In the second step the protein is applied across the middle of the gel and along the pH gradient. The net charge of the protein as a function of the pH is reflected in the mobility of the protein on electrophoresis perpendicular to the pH gradient. A 5% T, 3% C polyacrylamide gel (140 x 180 x 2 mm) containing 2.7% Pharmalytes 3-10 (Pharmacia) and 6 M urea (all chemicals were of electrophoresis purity) was cast between two glass plates, one of which was equipped with a plastic strip (1 x 2 x 105 mm) in order to retain a sample-trough in the gel. The pH gradient was developed at 1000 V for 2 h. A portion of 0.4 mg of cr-crystallin in 75 ~1 of a solution of 6 M urea was applied to the trough and electrophoresis in the second dimension was carried out at 640 V for 25 min. The protein was fixed in the gel for 1 h at 60°C using a solution of 1.5% sulphosalicylic acid, 5% trichloroacetic acid and 15% methanol in water. The protein bands were stained with 2% Coomassie Brilliant Blue R-250 in destaining solution consisting of 8% acetic acid and 25% ethanol in water. * Present address: Organon International, 5340 BH Oss, The Netherlands. 0021-9673/87/%03.50
0
Scientific Development
1987 Elsevier Science Publishers B.V.
Group, RPZIOR, PO Box 20,
Al
A2 Bl 82
Fig.
I. Electrophoretic
titration
curve of cx-crystallin
subunits
in 6 M urea
Fig. 1 shows the electrophoretic titration curve of the four ol-crystallin subunits in 6 M urea. The slope of the protein bands around the application site is proportional to dQ/dpH at the isoelectric point of the respective subunit, where Q is the net charge. Sluyterman and co-workers4*S presented some theoretical considerations on the difference between the pZ observed by chromatofocusing and the real pZ of a protein. Two negative terms contribute to this difference, one being proportional to the Donnan potential between the stationary and mobile phases and the other to the reciprocal of the Donnan potential and dQ/dpH. As dQ/dpH is negative, the sum of the two terms can become zero or positive at lower values of dQ/dpH. Apparently, with aB chains the two terms counterbalance each other, whereas the slope of the aA chains, which is a factor 0.85 smaller, is responsible for the 0.4 pH unit difference between the observed and real pZ of these subunits. ACKNOWLEDGEMENT
The authors thank Frans Biermans for technical assistance.
NOTES
REFERENCES 1 2 3 4 5
P. J. M. van den Oetelaar, R. Bezemer and H. J. Hoenders, _T.Chromntogr., 398 (1987) 323. H. Bloemendal and G. Groenewoud, Anal. Biochem., 117 (1981) 327. H. Michl, Munatsh. Chem., 83 (1952) 210. L. A. fE. Sluyterman and 0. Elgersma, J. Chromarogr., 150 (1978) 17. L. A. K. Sluyterman and J. Wijdenes, J. Chromatogr., 150 (1978) 31.
509
41 I (1987) 507-509
Elsevier Science Publishers B.V., Amsterdam ~ Printed in The Netherlands CHROM. 20 013
Note
Electrophoretic tein a-crystallin
titration
curve
in 6 A4 urea of the bovine
eye lens pro-
PIET J. M. VAN DEN OETELAAR* and HERMAN J. HOENDERS Department of Biochemistry, University of Ndjmegen, Kapittelweg 46,652s
EP Nij,egen
(The Netherlands)
(Received August 25th, 1987)
Recently we reported on the separation of the subunits of the bovine eye lens protein a-crystallin by means of fast protein liquid ehromatography-chromatofocusing in concentrated ureal. We noted that the pH at which the CYAN and aAz subunits eluted from the column differed by 0.41 pH unit from their pZ as determined by isoelectric focusing in concentrated urea, whereas there was no difference for the aB1 chain and only a difference of -0.09 pH unit for the ORBS subunit. Similar observations were made by Bloemendal and Groenewoud2 using a soft-gel PBE-94 column. We suggested that the difference in behaviour of the aA and aB chains is caused by a difference in the net charge of the subunits near their isoelectric point. The eleetrophoretic titration curve of a-crystallin is now presented in order to substantiate this assumption. Electrophoretic titration curves were introduced by Mich13 and have since proved useful for the determination of the optimal pH for other separation techniques such as chromatofocusing, ion-exchange chromatography and electrophoresis and for the detection of isozymes. The first step of the technique consists of the creation of a pH gradient in a horizontal slab gel using ampholytes. In the second step the protein is applied across the middle of the gel and along the pH gradient. The net charge of the protein as a function of the pH is reflected in the mobility of the protein on electrophoresis perpendicular to the pH gradient. A 5% T, 3% C polyacrylamide gel (140 x 180 x 2 mm) containing 2.7% Pharmalytes 3-10 (Pharmacia) and 6 M urea (all chemicals were of electrophoresis purity) was cast between two glass plates, one of which was equipped with a plastic strip (1 x 2 x 105 mm) in order to retain a sample-trough in the gel. The pH gradient was developed at 1000 V for 2 h. A portion of 0.4 mg of cr-crystallin in 75 ~1 of a solution of 6 M urea was applied to the trough and electrophoresis in the second dimension was carried out at 640 V for 25 min. The protein was fixed in the gel for 1 h at 60°C using a solution of 1.5% sulphosalicylic acid, 5% trichloroacetic acid and 15% methanol in water. The protein bands were stained with 2% Coomassie Brilliant Blue R-250 in destaining solution consisting of 8% acetic acid and 25% ethanol in water. * Present address: Organon International, 5340 BH Oss, The Netherlands. 0021-9673/87/%03.50
0
Scientific Development
1987 Elsevier Science Publishers B.V.
Group, RPZIOR, PO Box 20,
Al
A2 Bl 82
Fig.
I. Electrophoretic
titration
curve of cx-crystallin
subunits
in 6 M urea
Fig. 1 shows the electrophoretic titration curve of the four ol-crystallin subunits in 6 M urea. The slope of the protein bands around the application site is proportional to dQ/dpH at the isoelectric point of the respective subunit, where Q is the net charge. Sluyterman and co-workers4*S presented some theoretical considerations on the difference between the pZ observed by chromatofocusing and the real pZ of a protein. Two negative terms contribute to this difference, one being proportional to the Donnan potential between the stationary and mobile phases and the other to the reciprocal of the Donnan potential and dQ/dpH. As dQ/dpH is negative, the sum of the two terms can become zero or positive at lower values of dQ/dpH. Apparently, with aB chains the two terms counterbalance each other, whereas the slope of the aA chains, which is a factor 0.85 smaller, is responsible for the 0.4 pH unit difference between the observed and real pZ of these subunits. ACKNOWLEDGEMENT
The authors thank Frans Biermans for technical assistance.
NOTES
REFERENCES 1 2 3 4 5
P. J. M. van den Oetelaar, R. Bezemer and H. J. Hoenders, _T.Chromntogr., 398 (1987) 323. H. Bloemendal and G. Groenewoud, Anal. Biochem., 117 (1981) 327. H. Michl, Munatsh. Chem., 83 (1952) 210. L. A. fE. Sluyterman and 0. Elgersma, J. Chromarogr., 150 (1978) 17. L. A. K. Sluyterman and J. Wijdenes, J. Chromatogr., 150 (1978) 31.
509