Clinica Chimica Acta, 77 (1977) 9X-100 @ Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL
NOTE
CCA 8602
A SINGLE-STEP ISOLATION OF 011-ANTITRYPSIN FROM HUMAN SERUM USING DOUBLE-LAYER POLYACRYLAMIDE GEL ELECTROPHORESIS
WLADYSEAW
PAJDAK
and JAN SZNAJD
Department of Clinical Chemistry, Institute Kopernika 17, 31-501 Cracow (Poland) (Received
December
23rd,
of Internal
Medicine,
Medical Academy,
1976)
Introduction Serum glycoprotein, Q’I -AT is the major endogenous inhibitor of trypsin, chymotrypsin, elastase, plasmin, thrombin, collagenases, kallikrein and granulocyte proteinases [ 1,2]. In 1963 Laurel1 and Eriksson [3] first described a correlation between emphysema and inherited (Y,-AT deficiency. This has stimulated an interest in both the properties and function of this protein. During the past several years many methods for the isolation of LY,-AT have been described [1,4-71. However, the procedures used are multi-step, time consuming and very often result in obtaining o(, -AT in an at least partially inactive form. Besides, there is no satisfactory method for the isolation of this protein on a micro-scale. This report describes a simple technique for the quick isolation of human serum (Y,-AT using preparative electrophoresis in polyacrylamide gel. Materials and methods l-ml samples of human serum were obtained from donor blood. Twice crystallized trypsin (Serva) and BAEE (Sigma) were used for the determination of antitryptic activity [ 81. Anti-human serum, anti-albumin and anti-a, -AT sera (Behringwerke) were used for double immunodiffusion [9] and rocket immunoelectrophoresis [lo] tests. Analytical electrophoresis in polyacrylamide was performed according to Clarke [ll], the pH of the electrode buffer being 8.5 (Serva reagents were used for the gel, other reagents were of analytical grade). Preparative electrophoresis was performed as described before [ 121. The double layer (40 mm high) gel for preparative electrophoresis was prepared in a glass column of 30 mm internal diameter. The upper 20-mm half
99
consisted of 10% gel and the lower 20-mm half of 5% gel. One ml of serum dialyzed against 0.003 M NaCl with a trace amount (25 ~1, 0.01%) of bromophenol blue was put onto the gel. ~lectrophoresis was performed at a constant voltage of 200 V. During electrophoresis two bands of the dye, one with a sharply defined front, and the other diffused, marking the position of albumin, were seen. After the first band came to the bottom of the gel, fractions were collected every 2 min until the second colored band came out from the column. Each fraction was tested for antitrypsin activity and for tyl -AT and albumin, since albumin was found to be the only protein that may contaminate cxl -AT isolated by this procedure, owing to a close similarity of the molecular weights and isoelectric points of both proteins. For the sake of convenience, after a few preparative runs, the time intervals for the collection of (Y,-AT may be precisely determined. Generally, in the described conditions cy1-AT was collected as one Z-ml fraction between the two colored bands of the marking dye. Results and discussion In Fig. 1 the comparison of the patterns obtained when human serum was run on 5% and on double-layer 10%/S% polyacryl~ide gel is shown. It is clearly seen that the separation of Q, -AT from albumin is much better on double-layer gel, owing to the difference of the mobility of proteins in 10% and 5% gels: al-AT being eluted from the 5% gel before albumin was eluted from the 10% gel. When applied for preparative purposes this allows simplification of the procedure of a 1-AT isolation. The efficiency of the separation of (Y~-AT from albumin by preparative electrophoresis is shown in Fig. 2, and the tests for homogeneity of isolated 01~-AT are depicted in Fig. 3. By the criteria of immunoelectrophoresis and analytical
Fig. 1. Analytical separation of human serum on 5% polyacrylamide gel (left) and on double-layer lo%/ 5% polyacrylamide gel (right). Arrow indicates the boundary between 10% and 5% parts of the gel. Fig. 2. Rocket immunoelectrophoresis: nine fractions from preparative electrophoresis tested simultaneously for the presence of albumin and al-AT on separate immunoplates. Five fractions from the left are virtually free from albumin. The photograph was taken after putting both immunoplates together.
100
Fig.
3.
A.
acrylamide
Immunoelectrophoresis gel electrophoresis
of
isolated
of isolated
al-AT.
al-AT
the
compared
trough with
with
filled
whole
human
anlr-hugnan
wrunl.
13. Polv-
srrum.
polyacrylamide gel electrophoresis our preparation of a, -AT seems to be homogeneous. In conclusion, this method, permitting isolation of satisfactorily pure and biologically active cxl -AT from 1 ml of human serum in only 2 hours, offers a very easy ‘means for micro-scale preparation of this protein. Besides, the described “reversed double-layer gel system” may be of some use for other preparative purposes. Acknowledgements This work was supported in part by the Polish Academy of Sciences, PAN 337/VI. The authors are grateful for the technical assistance of Miss Graiyna Cierpikowska. References 1
Musiani,
P. and
2
Koj,
Chudzik,
3
Laurell.
A.,
C.B.
4
Liener,
5
Horng,
6
Pannell,
R.,
7
Wbitley,
E.J.
8
Homer,
9
Darcy,
I.E.,
Jr.. T.B.
J., Pajdak.
and
Eriksson,
Garrison,
W.J.
and
Gan.
Johnson,
G.M., D.A.
Tomasi,
and
10
Weeke,
B. (1973)
11
Clarke,
J.T.
12
Pajdak.
W.
(1964) (1973)
Clin. &and. Ann. Clin.
and
(1963) and
(1974)
D. and
Liener,
Katchman, (1972)
E.
O.R. J.C.
(1976)
W.
I.E.
Biochemistry
Dubin, &and.
Pravda. Texas
J. (1974) Prep.
B.J.
and
Zipf.
Acta
J. Immunol. Chim.
Acad. Acta
48,
Biochem.
Biol.
Med.
Invest. 32.
Biochemistry
Biochem. (1963)
2. Suppl. 121.
113-115
428436
Biophys. 15.
Biophys. 489-504 13,
Clin.
Chem.
5439-5445
1, 3746
9,
Acta
268,
199--206
132-140
5, 433-453
287-288
Vol. Sci.
(1973)
R.E.
37,
798-804
Biochim. Lab.
Z.
(1975)
15,
(1972)
J. Clin.
Rep.
Travis,
Chim. N.Y.
A.
28-43
Res.
Commun.
51,
436-443
BRIEF TECHNICAL
NOTE
CCA 8602
A SINGLE-STEP ISOLATION OF 011-ANTITRYPSIN FROM HUMAN SERUM USING DOUBLE-LAYER POLYACRYLAMIDE GEL ELECTROPHORESIS
WLADYSEAW
PAJDAK
and JAN SZNAJD
Department of Clinical Chemistry, Institute Kopernika 17, 31-501 Cracow (Poland) (Received
December
23rd,
of Internal
Medicine,
Medical Academy,
1976)
Introduction Serum glycoprotein, Q’I -AT is the major endogenous inhibitor of trypsin, chymotrypsin, elastase, plasmin, thrombin, collagenases, kallikrein and granulocyte proteinases [ 1,2]. In 1963 Laurel1 and Eriksson [3] first described a correlation between emphysema and inherited (Y,-AT deficiency. This has stimulated an interest in both the properties and function of this protein. During the past several years many methods for the isolation of LY,-AT have been described [1,4-71. However, the procedures used are multi-step, time consuming and very often result in obtaining o(, -AT in an at least partially inactive form. Besides, there is no satisfactory method for the isolation of this protein on a micro-scale. This report describes a simple technique for the quick isolation of human serum (Y,-AT using preparative electrophoresis in polyacrylamide gel. Materials and methods l-ml samples of human serum were obtained from donor blood. Twice crystallized trypsin (Serva) and BAEE (Sigma) were used for the determination of antitryptic activity [ 81. Anti-human serum, anti-albumin and anti-a, -AT sera (Behringwerke) were used for double immunodiffusion [9] and rocket immunoelectrophoresis [lo] tests. Analytical electrophoresis in polyacrylamide was performed according to Clarke [ll], the pH of the electrode buffer being 8.5 (Serva reagents were used for the gel, other reagents were of analytical grade). Preparative electrophoresis was performed as described before [ 121. The double layer (40 mm high) gel for preparative electrophoresis was prepared in a glass column of 30 mm internal diameter. The upper 20-mm half
99
consisted of 10% gel and the lower 20-mm half of 5% gel. One ml of serum dialyzed against 0.003 M NaCl with a trace amount (25 ~1, 0.01%) of bromophenol blue was put onto the gel. ~lectrophoresis was performed at a constant voltage of 200 V. During electrophoresis two bands of the dye, one with a sharply defined front, and the other diffused, marking the position of albumin, were seen. After the first band came to the bottom of the gel, fractions were collected every 2 min until the second colored band came out from the column. Each fraction was tested for antitrypsin activity and for tyl -AT and albumin, since albumin was found to be the only protein that may contaminate cxl -AT isolated by this procedure, owing to a close similarity of the molecular weights and isoelectric points of both proteins. For the sake of convenience, after a few preparative runs, the time intervals for the collection of (Y,-AT may be precisely determined. Generally, in the described conditions cy1-AT was collected as one Z-ml fraction between the two colored bands of the marking dye. Results and discussion In Fig. 1 the comparison of the patterns obtained when human serum was run on 5% and on double-layer 10%/S% polyacryl~ide gel is shown. It is clearly seen that the separation of Q, -AT from albumin is much better on double-layer gel, owing to the difference of the mobility of proteins in 10% and 5% gels: al-AT being eluted from the 5% gel before albumin was eluted from the 10% gel. When applied for preparative purposes this allows simplification of the procedure of a 1-AT isolation. The efficiency of the separation of (Y~-AT from albumin by preparative electrophoresis is shown in Fig. 2, and the tests for homogeneity of isolated 01~-AT are depicted in Fig. 3. By the criteria of immunoelectrophoresis and analytical
Fig. 1. Analytical separation of human serum on 5% polyacrylamide gel (left) and on double-layer lo%/ 5% polyacrylamide gel (right). Arrow indicates the boundary between 10% and 5% parts of the gel. Fig. 2. Rocket immunoelectrophoresis: nine fractions from preparative electrophoresis tested simultaneously for the presence of albumin and al-AT on separate immunoplates. Five fractions from the left are virtually free from albumin. The photograph was taken after putting both immunoplates together.
100
Fig.
3.
A.
acrylamide
Immunoelectrophoresis gel electrophoresis
of
isolated
of isolated
al-AT.
al-AT
the
compared
trough with
with
filled
whole
human
anlr-hugnan
wrunl.
13. Polv-
srrum.
polyacrylamide gel electrophoresis our preparation of a, -AT seems to be homogeneous. In conclusion, this method, permitting isolation of satisfactorily pure and biologically active cxl -AT from 1 ml of human serum in only 2 hours, offers a very easy ‘means for micro-scale preparation of this protein. Besides, the described “reversed double-layer gel system” may be of some use for other preparative purposes. Acknowledgements This work was supported in part by the Polish Academy of Sciences, PAN 337/VI. The authors are grateful for the technical assistance of Miss Graiyna Cierpikowska. References 1
Musiani,
P. and
2
Koj,
Chudzik,
3
Laurell.
A.,
C.B.
4
Liener,
5
Horng,
6
Pannell,
R.,
7
Wbitley,
E.J.
8
Homer,
9
Darcy,
I.E.,
Jr.. T.B.
J., Pajdak.
and
Eriksson,
Garrison,
W.J.
and
Gan.
Johnson,
G.M., D.A.
Tomasi,
and
10
Weeke,
B. (1973)
11
Clarke,
J.T.
12
Pajdak.
W.
(1964) (1973)
Clin. &and. Ann. Clin.
and
(1963) and
(1974)
D. and
Liener,
Katchman, (1972)
E.
O.R. J.C.
(1976)
W.
I.E.
Biochemistry
Dubin, &and.
Pravda. Texas
J. (1974) Prep.
B.J.
and
Zipf.
Acta
J. Immunol. Chim.
Acad. Acta
48,
Biochem.
Biol.
Med.
Invest. 32.
Biochemistry
Biochem. (1963)
2. Suppl. 121.
113-115
428436
Biophys. 15.
Biophys. 489-504 13,
Clin.
Chem.
5439-5445
1, 3746
9,
Acta
268,
199--206
132-140
5, 433-453
287-288
Vol. Sci.
(1973)
R.E.
37,
798-804
Biochim. Lab.
Z.
(1975)
15,
(1972)
J. Clin.
Rep.
Travis,
Chim. N.Y.
A.
28-43
Res.
Commun.
51,
436-443
