223
Clinica
Chimica
0 Elsevier
Acta,
Scientific
65 (1975) 223-226 Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7390
THE USE OF PREPARATIVE CONTINUOUS FLOW ELECTROPHORESIS TO STUDY LOW MOLECULAR WEIGHT PROTEINS IN URINE*
A. COLLIE?, C. TONNELLE,
M. LECLERCQ
and Y. MANUEL
Institut Pasteur de Lyon, Service de recherches sur les protkines et macromolPcules dans les liquides biologiques, 77, rue Pasteur, 69365 Lyon (Cedex 2) (France)
(Received
June 30, 1975)
Summary The authors propose a convenient method for the separation of low molecular weight proteins, present in tubular urines. In particular, the method is used for the purification of & microglobulin, after one recycling, and also for the isolation of the various post-y globulins which can later be separated on DEAE Sephadex.
Introduction In the last 10 years, the study of low molecular weight proteins in urine has grown in importance. Preparative continuous flow electrophoresis in free buffer film has shown itself to be an important stage in the isolation and purification of such proteins, in particular, the & microglobulin and post-y globulin gives a satisfac[l] . The use of the “Elphor VaP”, under specific conditions, tory yield of the highly purified proteins. Materials and method Urine samples These are taken from tubular dysfunction patients. Sodium azide 0.2 g/l is added as conserving agent. The urine samples, in which the protein concentration varies from 0.5 to 1 g/l, are then concentrated one hundred times through an Amicon UM2 filter.
* This work was supported by
contracts from INSERM.
224
Gel
fib-a tion
This is carried out using the urine of a single donor on Biogel P-100 with phosphate buffer pH 7.3. The fraction corresponding to the zone of molecular weight 10 OOO20 000 is then concentrated by dialysis against polyethylene glycol (PEG) (600 g/l) containing NaCl (9 g/l) to a final concentration of 10 g/l. This fraction is tested by Ouchterlony’s technique, to ensure the absence of serum proteins, such as albumin, transfer~n and hemopexin.
Preparative electrophoresis We use the “Elphor VaP” for preparative
continuous flow electrophoresis in free buffer film (Bender and Hobein, Munich) the whole operations being carried out at 4°C. The electrode buffer used is a Tris/EDTA/boric acid buffer (0.08 M/0.02 M/0.08 M) pH 8.3, the buffer flowing between the plates (chamber buffer) being diluted to one third. The fraction between molecular weight 10 000 and 20 000 was dialysed against the latter and injected at a rate of 2 ml/h. The separation is carried out at 100 mA and 2100 volts, the chamber buffer flowing at a rate of 70 ml/h. The fractions are collected in 48 tubes and the absorbance read at 280 nm in a Beckman Spectrophotometer. The protein concentration in the different tubes is estimated using the Amidoschwarz technique [2] and the concentration in the pure fractions measured by the biuret reaction [3] .
i
t
Fig. 1. Preparative
continuous
flow electrophoresis
in free buffer
film. Protein
profile
at 280
nm.
9m*e 3m i,o,wO.~~w:
1
7
3
4
5
6
7
r:
s
ID
Fig. 2. The 23 fractions tested on polyacwlamide Biogel P-100 M, 10 000-20 000.
1,
I2
13
agarose
‘,)
16
,B
17
18
9
2”
2,
gel. On the left, the fraction
21
2,
obtained
from
Each fraction is then concentrated by dialysis against PEG GO%/NaCl 0.9%. The quality of the separation is verified by electrophoresis in polyacrylamide agarose gel at 5% [4]. According to the degree of purity, the fractions are recycled using the same process. Results and discussion The first separation was divided into 23 fractions. Fraction sists of 5 tubes, is found to be rich in retinol-binding protein.
Fig. 3. Recycling of fraction 9, giving a pure fll microglobulin. on the right, normal human serum control.
4, which con-
On the left, unseparated
tubular
urine;
226
Fractions 8, 9 and 10 are very rich in p2 microglobulin. A single recycling of fraction 8 gave a highly purified pZ microglobulin. Fractions 14 to 20 contain the different post-y globulins, which can then be separated on DEAE-Sephadex. By this method we were able to eliminate contamination by lysozyme in urine which is found in fractions 21-23. The different separations carried out on the “Elphor Vap” have always given a yield of about 70%, which is of great interest. It is important to point out that the good results obtained are mostly due to the rigorous control of the fractions carried out on Biogel P-100 preceding the electrophoresis. This ensured the absence of any serum contaminants of identical electrophoretic mobility to those proteins in the fraction which we wanted to purify. Therefore to conclude: by the appropriate choice of tubular urines, rich in certain proteins, we can isolate the pure protein after one gel filtration and this giving reproducible results and one or two preparative electrophoreses, a satisfactory yield. References A. and Tonnelle, C. (1973) Protides 1 Manuel, Y., Leclercq, M.. Co&, Colloquium Brugge (Peeters, H., ed.), p. 505 2 Groulade. J. and Groslambert, P. (1960) Ann. Biol. Clin. 18. 595 3 Goa, J. (1953) Stand. J. Clin. Lab. Invest. 5, 218 4 Uriel, J. (1966) Bull. Sm. Chin Biol. 48,969
of the
Biological
Fluids,
21st
Clinica
Chimica
0 Elsevier
Acta,
Scientific
65 (1975) 223-226 Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7390
THE USE OF PREPARATIVE CONTINUOUS FLOW ELECTROPHORESIS TO STUDY LOW MOLECULAR WEIGHT PROTEINS IN URINE*
A. COLLIE?, C. TONNELLE,
M. LECLERCQ
and Y. MANUEL
Institut Pasteur de Lyon, Service de recherches sur les protkines et macromolPcules dans les liquides biologiques, 77, rue Pasteur, 69365 Lyon (Cedex 2) (France)
(Received
June 30, 1975)
Summary The authors propose a convenient method for the separation of low molecular weight proteins, present in tubular urines. In particular, the method is used for the purification of & microglobulin, after one recycling, and also for the isolation of the various post-y globulins which can later be separated on DEAE Sephadex.
Introduction In the last 10 years, the study of low molecular weight proteins in urine has grown in importance. Preparative continuous flow electrophoresis in free buffer film has shown itself to be an important stage in the isolation and purification of such proteins, in particular, the & microglobulin and post-y globulin gives a satisfac[l] . The use of the “Elphor VaP”, under specific conditions, tory yield of the highly purified proteins. Materials and method Urine samples These are taken from tubular dysfunction patients. Sodium azide 0.2 g/l is added as conserving agent. The urine samples, in which the protein concentration varies from 0.5 to 1 g/l, are then concentrated one hundred times through an Amicon UM2 filter.
* This work was supported by
contracts from INSERM.
224
Gel
fib-a tion
This is carried out using the urine of a single donor on Biogel P-100 with phosphate buffer pH 7.3. The fraction corresponding to the zone of molecular weight 10 OOO20 000 is then concentrated by dialysis against polyethylene glycol (PEG) (600 g/l) containing NaCl (9 g/l) to a final concentration of 10 g/l. This fraction is tested by Ouchterlony’s technique, to ensure the absence of serum proteins, such as albumin, transfer~n and hemopexin.
Preparative electrophoresis We use the “Elphor VaP” for preparative
continuous flow electrophoresis in free buffer film (Bender and Hobein, Munich) the whole operations being carried out at 4°C. The electrode buffer used is a Tris/EDTA/boric acid buffer (0.08 M/0.02 M/0.08 M) pH 8.3, the buffer flowing between the plates (chamber buffer) being diluted to one third. The fraction between molecular weight 10 000 and 20 000 was dialysed against the latter and injected at a rate of 2 ml/h. The separation is carried out at 100 mA and 2100 volts, the chamber buffer flowing at a rate of 70 ml/h. The fractions are collected in 48 tubes and the absorbance read at 280 nm in a Beckman Spectrophotometer. The protein concentration in the different tubes is estimated using the Amidoschwarz technique [2] and the concentration in the pure fractions measured by the biuret reaction [3] .
i
t
Fig. 1. Preparative
continuous
flow electrophoresis
in free buffer
film. Protein
profile
at 280
nm.
9m*e 3m i,o,wO.~~w:
1
7
3
4
5
6
7
r:
s
ID
Fig. 2. The 23 fractions tested on polyacwlamide Biogel P-100 M, 10 000-20 000.
1,
I2
13
agarose
‘,)
16
,B
17
18
9
2”
2,
gel. On the left, the fraction
21
2,
obtained
from
Each fraction is then concentrated by dialysis against PEG GO%/NaCl 0.9%. The quality of the separation is verified by electrophoresis in polyacrylamide agarose gel at 5% [4]. According to the degree of purity, the fractions are recycled using the same process. Results and discussion The first separation was divided into 23 fractions. Fraction sists of 5 tubes, is found to be rich in retinol-binding protein.
Fig. 3. Recycling of fraction 9, giving a pure fll microglobulin. on the right, normal human serum control.
4, which con-
On the left, unseparated
tubular
urine;
226
Fractions 8, 9 and 10 are very rich in p2 microglobulin. A single recycling of fraction 8 gave a highly purified pZ microglobulin. Fractions 14 to 20 contain the different post-y globulins, which can then be separated on DEAE-Sephadex. By this method we were able to eliminate contamination by lysozyme in urine which is found in fractions 21-23. The different separations carried out on the “Elphor Vap” have always given a yield of about 70%, which is of great interest. It is important to point out that the good results obtained are mostly due to the rigorous control of the fractions carried out on Biogel P-100 preceding the electrophoresis. This ensured the absence of any serum contaminants of identical electrophoretic mobility to those proteins in the fraction which we wanted to purify. Therefore to conclude: by the appropriate choice of tubular urines, rich in certain proteins, we can isolate the pure protein after one gel filtration and this giving reproducible results and one or two preparative electrophoreses, a satisfactory yield. References A. and Tonnelle, C. (1973) Protides 1 Manuel, Y., Leclercq, M.. Co&, Colloquium Brugge (Peeters, H., ed.), p. 505 2 Groulade. J. and Groslambert, P. (1960) Ann. Biol. Clin. 18. 595 3 Goa, J. (1953) Stand. J. Clin. Lab. Invest. 5, 218 4 Uriel, J. (1966) Bull. Sm. Chin Biol. 48,969
of the
Biological
Fluids,
21st
