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Heterogeneity in electrophoretic mobility and in molecular size of lactate dehydrogenase-immunoglobulin G complexes
149
Clinica
Chimica
Acta,
76 (1977)
149-153 Biomedical Press
@ Elsevier/North-Holland
CCA 8463
HETEROGENEITY IN ELECTROPHORETIC MOBILITY AND IN MOLECULAR SIZE OF LACTATE DEHYDROGENASE-IMMUNOGLOBULIN G COMPLEXES
JEIKE
BIEWENGA
Department
(Received
of Histology,
November
Free
University,
P.O. Box
7161,
Amsterdam
(The
Netherlands)
22nd, 1976)
summary
Some human sera have been found to contain lactate dehydrogenase (LDH)immunoglobulin complexes. A serum with LDH-IgG3 complexes was further analysed. Complexes with different net charge and different molecular size were found. Their isoelectric points, measured by isoelectric focusing, were in the range of pH 6.3 to 7.2. Gel-filtration experiments showed that the molecular size varied from about 300 000 to 1 000 000. Most of the complexes could be precipitated from solution with 30-40% saturated ammonium sulfate or at low ionic strength.
Introduction In some human sera lactate dehydrogenase-immunoglobulin complexes occur. Cases with lactate dehydrogenase-immunoglobulin A (LDH-IgA) as well as with lactate dehydrogenase-immunoglobulin G (LDH-IgG) complexes have been described [l-l. The sera containing such complexes are characterized by abnormal electrophoretic mobility of LDH, compared to that of normal LDH isoenzymes. The anomaly consists of an abnormal location of LDH and in the majority of the cases the complexes also give rise to more diffuse bands. The LDH-IgG3 complexes of one patient were studied in more detail. It is shown that they were precipitated at low ionic strength and were heterogeneous with respect to both electrophoretic mobility and molecular weight. Materials and methods The serum studied was obtained from patient severe lung function disorders [6]. LDH isoenzymes mobility were not observed in his serum.
Vr, aged 70 years. He had with normal electrophoretic
150
Ammonium sulfate fractionation was performed by the addition of a saturated solution of (NH4)#04 to the serum or the subsequent supernatants. After the addition of ammonium sulfate the mixtures were kept at 4°C for at least 2.5 hours before centrifugation. The sediments obtained at 30%, 40% and 50% ammonium sulfate saturation were dissolved in and dialysed against demineralized water. New precipitates formed during the dialysis were redissolved in a small volume of 0.1 M phosphate buffer, pH 7.4. Gel-filtration experiments were performed on Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden) or Ultrogel AcA 22 (LKB Produkter AB, Bromma, Sweden) columns at 4°C according to the manufacturer’s prescriptions. A large sample volume was chosen in order to be able to determine the LDH activity in the separate eluate fractions. If necessary for electrophoretic analysis eluate fractions were concentrated on Minicon filters (Amicon, Oosterhout, The Netherlands). Isoelectric focusing of serum Vr was performed in polyacrylamide gel using a pH gradient from 3.5 to 9.5 [7]. Results The electrophoretic pattern of the LDH of serum Vr on agar gel is shown in Fig. 1. On isoelectric focusing of the serum the complexes focused in the pH region of 6.3 to 7.2 as was demonstrated by staining the gel for LDH activity. Some sharp bands were observed in an otherwise diffusely stained area. On (NH4)I?SOd fractionation the LDH activity of this serum was partly precipitated at 30% ammonium sulfate saturation; at 40% saturation the precipitation of LDH activity was nearly complete. The greater part of the LDH activity appeared to be insoluble in demineralized water but could be dissolved in phosphate buffer (Figs. 2 and 3). In addition to the very diffuse LDH band, situated between normal LDH-3 and LDH-4, a weaker LDH band was observed between normal LDH-4 and LDH-5 (Figs. 2 and 3). The latter band was hardly visible in the LDH pattern of the serum (Fig. 1). On gel-filtration of the serum on Sephadex G-200 most of the LDH activity
+
a control
+
serum control serum
b
serum Vr Fig
Fig. tions
1. Electrophoretic
2.
Electrophoretic
from:
a, 30%:
pattern
patterns b, 40%
of the
LDH
of the
and c. 50%
of swum
LDH
C Vr on agar gel.
of the supernatants
(NHq)zS04
saturation
obtained of serum
on dialysis Vr.
of the
sediment
frac-
151
4280 t
------
LDH actlvlty
4.0 -
+
control serum 2.0-
a
-
b
C
160
200
260ml
240
-eluate
Fig. 3. Electrophoretic patterns of the LDH of the resoluhilized sediments obtained on dialysis sediment fractions from: a, 30%; b. 40% and c, 50% (NHq)zS04 saturation of serum Vr. Fig. 4. Elution pattern of serum Vr obtained by gel-filtration Buffer solution: 0.1 M Tris/HCl. 0.5 M NaCl, pH 8.0. Sample
on Sephadex G-ZOO. Column: volume: 5 ml.
volume
ot tnr
97 X 2.5 cm.
was eluted in the void volume. The LDH peak was broadened towards the second protein peak (Fig. 4). On Ultrogel AcA 22 the LDH activity of the serum was eluted in three separate peaks (Fig. 5). The eluate fractions of the Ultrogel column containing LDH activity were collected in four pools as indicated (Fig. 5). After concentration they were examined for their LDH and protein patterns (Fig. 6). Electrophoresis of a small volume of each of the concentrated pools mixed
-~~~---LDH activity
400
600
Fig. 5. Elution pattern of serum Buffer solution: 0.1 M phosphate,
800
1000 ml
beluate volume Vr obtained by gel-filtration on Ultrogel AcA 22. Column: 0.5 M N&l, pH 8.0. Sample volume: 5 ml.
91 X 2.6 cm.
152
pool II
pool III
pool IV control serum
b
c
+
+
control serum
J
control serum pool I
pool I
P pool II
pool II
pool III
pool III
-*
* pool IV’ control Serum Fig. AcA
6.
Electrophoretic
22.
antiserum
a, to
agar whole
gel
patterns electrophoresis. human
serum;
of
the
pooled stained
stained
for
control serum
0
fractions for
pool IV
0 -““’
LDH
proteins
obtained activity. (b)
and
on
gel-filtration
b and LDH
of
serum
c. imlnunoelectrophoresis
activity
Vr
on
Ultrogel with
an
(c).
with an equal volume of a control serum containing all normal LDH isoenzymes, was used to test whether binding of normal LDH occurred. Pools II, III and IV were found to bind all normal LDH isoenzymes; pool I did not bind the normal LDH isoenzymes [ 2,6 1. Discussion A human serum containing soluble complexes of LDH and IgG3 was studied. These complexes were precipitated with 30-405X saturated ammonium sulfate and were insoluble in demineralized water. The molecular weight of the complexes estimated from gel-filtration on Ultrogel AcA 22 is about 1 000 000 for the largest complexes and goes down to 300 000-400 000 for the complexes with the lower molecular weight. The higher value is estimated from the fact that LDH activity was already eluted from the Ultrogel AcA 22 column at the beginning of the elution of the IgM fraction (mol.wt. of IgM is 900 000). The
153
lower value is based on the elution of the smaller complexes before IgG on Ultrogel AcA 22 as well as on Sephadex G-200. Comparable values had been obtained by polyacrylamide-gradient gel electrophoresis of serum Vr (unpublished). Since suitable calibration proteins are not available for that method in this high molecular weight region, exact values could not be obtained. The LDH fraction which was found between normal LDH-4 and LDH-5 on agar gel electrophoresis (Figs. 2 and 3), probably belongs to the lower molecular weight complexes, since similar electrophoretic mobility is found among the smaller complexes of pools III and IV (Fig. 6) and this LDH fraction. The largest complexes (pool I and II, Fig. 6a) have an electrophoretic mobility between that of normal LDH-3 and LDH-4. On the basis of these observations and the results of the isoelectric focusing experiments it is evident that the LDH-IgG3 complexes are heterogeneous both with respect to electrophoretic mobility (differences in isoelectric point) and to molecular weight. The largest complexes of pool I are saturated with LDH since LDH was not bound by this pool. The other complexes (pools II-IV) are perhaps not saturated with LDH but the binding of LDH by these pools can also be explained from the presence of LDH binding sites on free IgG3 molecules. The differences in shape of the LDH staining in the immunoelectrophoretic patterns of the four pools (Fig. 6c) is attributed to differences in the concentration of IgG. When an excess of IgG is present (pools III and IV and also in the serum) the formation of the IgG precipitation line prevents passage of antibodies to IgG through this precipitation line. Consequently, no LDH-IgG, complexes precipitate at a greater distance from the trough than this IgG line. When only minor amounts of IgG are present (pools I and II), antibodies to IgG precipitate the LDH-IgG3 complexes at a greater distance from the trough. The precipitation became diffuse in these cases. The latter observation must be due to the formation of an immune precipitate that, once formed with the already large LDH-IgG3 complexes, is insoluble in excess of antiserum. The normal process of precipitation and resolubilization until a sharp precipitation line is formed, is supposed to be disturbed. Acknowledgement The author is grateful to Peter Kreuger for her technical assistance. The serum samples were obtained from Dr. H.B.A. Hellendoorn and Dr. J.L. de Graaff. Prof. Dr. H.L. Langevoort was helpful in preparing the manuscript. References 1
Kindmark.
2
Biewenga,
C.O.
(1969)
Stand.
3
Biewenga,
4
Markel.
5
Biewenga.
J. and
Feltkamp.
T.E.W.
(1975)
Clin.
Chim.
Acta
58,
239-249
6
Biewenga,
J. and
Feltkamp.
T.E.W.
(1975)
Clin.
Chim.
Acta
64.
101-116
7
Karlsson.
J. (1972)
Clin.
J. (1973)
Clin.
S.F.
C..
and Janich.
Davies.
H.,
J. Clin.
Chim.
Acta
Chim.
Acta
S.L.
(1974)
Ghman,
Invest.
24,
40.
407-414
47,
139-147
Am.
J. and
J. Clin.
Anderson,
49-53
Pathol.
61,
U.-B.
328-332
(1973)
LKB
Application
Note
No.
75
Clinica
Chimica
Acta,
76 (1977)
149-153 Biomedical Press
@ Elsevier/North-Holland
CCA 8463
HETEROGENEITY IN ELECTROPHORETIC MOBILITY AND IN MOLECULAR SIZE OF LACTATE DEHYDROGENASE-IMMUNOGLOBULIN G COMPLEXES
JEIKE
BIEWENGA
Department
(Received
of Histology,
November
Free
University,
P.O. Box
7161,
Amsterdam
(The
Netherlands)
22nd, 1976)
summary
Some human sera have been found to contain lactate dehydrogenase (LDH)immunoglobulin complexes. A serum with LDH-IgG3 complexes was further analysed. Complexes with different net charge and different molecular size were found. Their isoelectric points, measured by isoelectric focusing, were in the range of pH 6.3 to 7.2. Gel-filtration experiments showed that the molecular size varied from about 300 000 to 1 000 000. Most of the complexes could be precipitated from solution with 30-40% saturated ammonium sulfate or at low ionic strength.
Introduction In some human sera lactate dehydrogenase-immunoglobulin complexes occur. Cases with lactate dehydrogenase-immunoglobulin A (LDH-IgA) as well as with lactate dehydrogenase-immunoglobulin G (LDH-IgG) complexes have been described [l-l. The sera containing such complexes are characterized by abnormal electrophoretic mobility of LDH, compared to that of normal LDH isoenzymes. The anomaly consists of an abnormal location of LDH and in the majority of the cases the complexes also give rise to more diffuse bands. The LDH-IgG3 complexes of one patient were studied in more detail. It is shown that they were precipitated at low ionic strength and were heterogeneous with respect to both electrophoretic mobility and molecular weight. Materials and methods The serum studied was obtained from patient severe lung function disorders [6]. LDH isoenzymes mobility were not observed in his serum.
Vr, aged 70 years. He had with normal electrophoretic
150
Ammonium sulfate fractionation was performed by the addition of a saturated solution of (NH4)#04 to the serum or the subsequent supernatants. After the addition of ammonium sulfate the mixtures were kept at 4°C for at least 2.5 hours before centrifugation. The sediments obtained at 30%, 40% and 50% ammonium sulfate saturation were dissolved in and dialysed against demineralized water. New precipitates formed during the dialysis were redissolved in a small volume of 0.1 M phosphate buffer, pH 7.4. Gel-filtration experiments were performed on Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden) or Ultrogel AcA 22 (LKB Produkter AB, Bromma, Sweden) columns at 4°C according to the manufacturer’s prescriptions. A large sample volume was chosen in order to be able to determine the LDH activity in the separate eluate fractions. If necessary for electrophoretic analysis eluate fractions were concentrated on Minicon filters (Amicon, Oosterhout, The Netherlands). Isoelectric focusing of serum Vr was performed in polyacrylamide gel using a pH gradient from 3.5 to 9.5 [7]. Results The electrophoretic pattern of the LDH of serum Vr on agar gel is shown in Fig. 1. On isoelectric focusing of the serum the complexes focused in the pH region of 6.3 to 7.2 as was demonstrated by staining the gel for LDH activity. Some sharp bands were observed in an otherwise diffusely stained area. On (NH4)I?SOd fractionation the LDH activity of this serum was partly precipitated at 30% ammonium sulfate saturation; at 40% saturation the precipitation of LDH activity was nearly complete. The greater part of the LDH activity appeared to be insoluble in demineralized water but could be dissolved in phosphate buffer (Figs. 2 and 3). In addition to the very diffuse LDH band, situated between normal LDH-3 and LDH-4, a weaker LDH band was observed between normal LDH-4 and LDH-5 (Figs. 2 and 3). The latter band was hardly visible in the LDH pattern of the serum (Fig. 1). On gel-filtration of the serum on Sephadex G-200 most of the LDH activity
+
a control
+
serum control serum
b
serum Vr Fig
Fig. tions
1. Electrophoretic
2.
Electrophoretic
from:
a, 30%:
pattern
patterns b, 40%
of the
LDH
of the
and c. 50%
of swum
LDH
C Vr on agar gel.
of the supernatants
(NHq)zS04
saturation
obtained of serum
on dialysis Vr.
of the
sediment
frac-
151
4280 t
------
LDH actlvlty
4.0 -
+
control serum 2.0-
a
-
b
C
160
200
260ml
240
-eluate
Fig. 3. Electrophoretic patterns of the LDH of the resoluhilized sediments obtained on dialysis sediment fractions from: a, 30%; b. 40% and c, 50% (NHq)zS04 saturation of serum Vr. Fig. 4. Elution pattern of serum Vr obtained by gel-filtration Buffer solution: 0.1 M Tris/HCl. 0.5 M NaCl, pH 8.0. Sample
on Sephadex G-ZOO. Column: volume: 5 ml.
volume
ot tnr
97 X 2.5 cm.
was eluted in the void volume. The LDH peak was broadened towards the second protein peak (Fig. 4). On Ultrogel AcA 22 the LDH activity of the serum was eluted in three separate peaks (Fig. 5). The eluate fractions of the Ultrogel column containing LDH activity were collected in four pools as indicated (Fig. 5). After concentration they were examined for their LDH and protein patterns (Fig. 6). Electrophoresis of a small volume of each of the concentrated pools mixed
-~~~---LDH activity
400
600
Fig. 5. Elution pattern of serum Buffer solution: 0.1 M phosphate,
800
1000 ml
beluate volume Vr obtained by gel-filtration on Ultrogel AcA 22. Column: 0.5 M N&l, pH 8.0. Sample volume: 5 ml.
91 X 2.6 cm.
152
pool II
pool III
pool IV control serum
b
c
+
+
control serum
J
control serum pool I
pool I
P pool II
pool II
pool III
pool III
-*
* pool IV’ control Serum Fig. AcA
6.
Electrophoretic
22.
antiserum
a, to
agar whole
gel
patterns electrophoresis. human
serum;
of
the
pooled stained
stained
for
control serum
0
fractions for
pool IV
0 -““’
LDH
proteins
obtained activity. (b)
and
on
gel-filtration
b and LDH
of
serum
c. imlnunoelectrophoresis
activity
Vr
on
Ultrogel with
an
(c).
with an equal volume of a control serum containing all normal LDH isoenzymes, was used to test whether binding of normal LDH occurred. Pools II, III and IV were found to bind all normal LDH isoenzymes; pool I did not bind the normal LDH isoenzymes [ 2,6 1. Discussion A human serum containing soluble complexes of LDH and IgG3 was studied. These complexes were precipitated with 30-405X saturated ammonium sulfate and were insoluble in demineralized water. The molecular weight of the complexes estimated from gel-filtration on Ultrogel AcA 22 is about 1 000 000 for the largest complexes and goes down to 300 000-400 000 for the complexes with the lower molecular weight. The higher value is estimated from the fact that LDH activity was already eluted from the Ultrogel AcA 22 column at the beginning of the elution of the IgM fraction (mol.wt. of IgM is 900 000). The
153
lower value is based on the elution of the smaller complexes before IgG on Ultrogel AcA 22 as well as on Sephadex G-200. Comparable values had been obtained by polyacrylamide-gradient gel electrophoresis of serum Vr (unpublished). Since suitable calibration proteins are not available for that method in this high molecular weight region, exact values could not be obtained. The LDH fraction which was found between normal LDH-4 and LDH-5 on agar gel electrophoresis (Figs. 2 and 3), probably belongs to the lower molecular weight complexes, since similar electrophoretic mobility is found among the smaller complexes of pools III and IV (Fig. 6) and this LDH fraction. The largest complexes (pool I and II, Fig. 6a) have an electrophoretic mobility between that of normal LDH-3 and LDH-4. On the basis of these observations and the results of the isoelectric focusing experiments it is evident that the LDH-IgG3 complexes are heterogeneous both with respect to electrophoretic mobility (differences in isoelectric point) and to molecular weight. The largest complexes of pool I are saturated with LDH since LDH was not bound by this pool. The other complexes (pools II-IV) are perhaps not saturated with LDH but the binding of LDH by these pools can also be explained from the presence of LDH binding sites on free IgG3 molecules. The differences in shape of the LDH staining in the immunoelectrophoretic patterns of the four pools (Fig. 6c) is attributed to differences in the concentration of IgG. When an excess of IgG is present (pools III and IV and also in the serum) the formation of the IgG precipitation line prevents passage of antibodies to IgG through this precipitation line. Consequently, no LDH-IgG, complexes precipitate at a greater distance from the trough than this IgG line. When only minor amounts of IgG are present (pools I and II), antibodies to IgG precipitate the LDH-IgG3 complexes at a greater distance from the trough. The precipitation became diffuse in these cases. The latter observation must be due to the formation of an immune precipitate that, once formed with the already large LDH-IgG3 complexes, is insoluble in excess of antiserum. The normal process of precipitation and resolubilization until a sharp precipitation line is formed, is supposed to be disturbed. Acknowledgement The author is grateful to Peter Kreuger for her technical assistance. The serum samples were obtained from Dr. H.B.A. Hellendoorn and Dr. J.L. de Graaff. Prof. Dr. H.L. Langevoort was helpful in preparing the manuscript. References 1
Kindmark.
2
Biewenga,
C.O.
(1969)
Stand.
3
Biewenga,
4
Markel.
5
Biewenga.
J. and
Feltkamp.
T.E.W.
(1975)
Clin.
Chim.
Acta
58,
239-249
6
Biewenga,
J. and
Feltkamp.
T.E.W.
(1975)
Clin.
Chim.
Acta
64.
101-116
7
Karlsson.
J. (1972)
Clin.
J. (1973)
Clin.
S.F.
C..
and Janich.
Davies.
H.,
J. Clin.
Chim.
Acta
Chim.
Acta
S.L.
(1974)
Ghman,
Invest.
24,
40.
407-414
47,
139-147
Am.
J. and
J. Clin.
Anderson,
49-53
Pathol.
61,
U.-B.
328-332
(1973)
LKB
Application
Note
No.
75