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Electrophoretic heterogeneity of α2-macroglobulin
SHORT COMMUNICATIONS
Electrophoretic
137
heterogeneity of a,-macroglobulin
Schonenberger, Smidtberger and Schultze l isolated human a,-macroglobulin from serum and found its molecular weight, calculated from sedimentation and diffusion data, to be 82oooo~50 ooo. On centrifugation and on free electrophoresis the substance appeared homogeneous. On starch gel electrophoresis or,-macroglobulin was identified as the slow-migrating distinct band previously called See,. The Sa, band may sometimes appear to be split but it has not been proved whether both fractions represent a,-macroglobulin. On electrophoretic separation of human sera in cooled agarose we have regularly observed that the ~~-macroglobulin fraction spreads more than a compound with a homogeneous charge and low diffusion rate. The distribution of the oc,-macroglobulin molecules along the electrophoretic ol+one is estimated by cutting out a longitudinal strip of the gel in the direction of migration containing the a,- to /I-globulins and by running these Ix,-globulins into another agarose gel containing rabbit anti-cc,-macroglobulin (antigen-antibody crossed electrophoresis) 2 after turning the electric field 90”. With this technique electrophor~t~cally homogeneous proteins give symmetric peaks of antigen-antibody precipitates with their bases resting on the insertion line. Fig. I gives examples of the precipitation patterns produced by ol,-macroglobulin in different sera. The maximum height of each peak corresponds to the frontier of the electrophoretic a,-zone seen in sera belonging to haptoglobin type Z-I or 2-2 with the barbital buffer used (pH 8.6, 0.07 IM with z mN calcium lactate). The electrophoretie mobility of the fastest as well as the slowest subfraction of ~~-macroglob~in seems to be roughly constant in all sera. But the partition of a,-macroglobulin molecules with fast, medium or slow migration rate varies from serum to serum, especially during disease. The varying migration rate is not caused by buffer-ion interaction or
Fig. I. Antigen-antibody crossed electrophoresis, run under identical conditions, a specific anti-a,-macroglobulin serum. The migration in the first electrophoretic towards the left. Ch%.
Chim.
Acta,
of six sera with separation was
14 (1966)
137-138
SHORT COMMUNICATIONS
138
by trailing, since the molecules within the different parts of the cr,-macroglobulin zone retain their migration rate when the cr,-zone of the gel is cut into transverse sections and the fractions are re-run (Fig. 2). Treatment of serum with highly active, purified pneumococcal neuraminidase causes no demonstrable change in degree of
Fig. 2. Antigenantibody crossed elcctrophoresis of pooled human serum (left) and its electrophoretic fractions (right). The fractions were obtained by cutting the ccz-zone of an agarose gel (after elcctrophoresis of the serum) into narrow strips (2 mm) according to the left figure. The electrophoresis was run towards the left; the vertical line in the peaks indicates 50 mm distance of migration (albumin front ioo mm)
electrophoretic heterogeneity or in mean mobility, while the mobility for other plasma proteins is reduced in the typical way. Differences in acetylneuraminic acid content can thus be excluded as a probable cause of the heterogeneity. It seems more reasonable that the heterogeneity depends on formation of complexes between tL,-macroglobulin and other compounds since it has been shown that cr,-macroglobulin firmly links substances like trypsinz, somatotropin 4, plasmin and insulina*7. This work was supported No. 13X-581-01). Department of Clinical Chemistry, Malmb’ General Hospital, University of Lund (Sweden)
by the Swedish Medical Research
Council (Project
P. 0. C~ANROT C.-B. LAURELL
I M. SCHBNENBERGER,R.SCHMIDTBERGERANDH.E.SCHULTZE,Z. Naturforsch.,13 b(rg58)761. 2 C. B. LAURELL, Anal. Biochem., IO (1965) 358. 3 J. W. MEHL, W. O'CONNELL AND J. DEGROOT, Science, 145 (1964) 821. 4 D. R. HADDEN AND T. E. PROUT, Nature, 202 (1964) 1342. 5 H.E. SCHULTZE,N. HEIMBURGER, K. HEIDE, H.HAUPT, K. ST~RIKO AND G. SCHWICK,PYOC. 9th Europ. Sot. Haematol., Lisbon, 1963. Karger, Bawl/New York, 1963, p. 1315. 6 J. CLAUSEN, F. GJEDDE AND K. JORGENSEN,Proc. Sot. Exptl. Biol. Med., 112 (1963) 778. 7 G. R. ZAHND AND J. J. SCHEIDEGGER, Helv. Med. Acta, 30 (1963) 506.
Received Clin. Chim.
January zest, 1966 Acta,
14 (1966) 137-138
Electrophoretic
137
heterogeneity of a,-macroglobulin
Schonenberger, Smidtberger and Schultze l isolated human a,-macroglobulin from serum and found its molecular weight, calculated from sedimentation and diffusion data, to be 82oooo~50 ooo. On centrifugation and on free electrophoresis the substance appeared homogeneous. On starch gel electrophoresis or,-macroglobulin was identified as the slow-migrating distinct band previously called See,. The Sa, band may sometimes appear to be split but it has not been proved whether both fractions represent a,-macroglobulin. On electrophoretic separation of human sera in cooled agarose we have regularly observed that the ~~-macroglobulin fraction spreads more than a compound with a homogeneous charge and low diffusion rate. The distribution of the oc,-macroglobulin molecules along the electrophoretic ol+one is estimated by cutting out a longitudinal strip of the gel in the direction of migration containing the a,- to /I-globulins and by running these Ix,-globulins into another agarose gel containing rabbit anti-cc,-macroglobulin (antigen-antibody crossed electrophoresis) 2 after turning the electric field 90”. With this technique electrophor~t~cally homogeneous proteins give symmetric peaks of antigen-antibody precipitates with their bases resting on the insertion line. Fig. I gives examples of the precipitation patterns produced by ol,-macroglobulin in different sera. The maximum height of each peak corresponds to the frontier of the electrophoretic a,-zone seen in sera belonging to haptoglobin type Z-I or 2-2 with the barbital buffer used (pH 8.6, 0.07 IM with z mN calcium lactate). The electrophoretie mobility of the fastest as well as the slowest subfraction of ~~-macroglob~in seems to be roughly constant in all sera. But the partition of a,-macroglobulin molecules with fast, medium or slow migration rate varies from serum to serum, especially during disease. The varying migration rate is not caused by buffer-ion interaction or
Fig. I. Antigen-antibody crossed electrophoresis, run under identical conditions, a specific anti-a,-macroglobulin serum. The migration in the first electrophoretic towards the left. Ch%.
Chim.
Acta,
of six sera with separation was
14 (1966)
137-138
SHORT COMMUNICATIONS
138
by trailing, since the molecules within the different parts of the cr,-macroglobulin zone retain their migration rate when the cr,-zone of the gel is cut into transverse sections and the fractions are re-run (Fig. 2). Treatment of serum with highly active, purified pneumococcal neuraminidase causes no demonstrable change in degree of
Fig. 2. Antigenantibody crossed elcctrophoresis of pooled human serum (left) and its electrophoretic fractions (right). The fractions were obtained by cutting the ccz-zone of an agarose gel (after elcctrophoresis of the serum) into narrow strips (2 mm) according to the left figure. The electrophoresis was run towards the left; the vertical line in the peaks indicates 50 mm distance of migration (albumin front ioo mm)
electrophoretic heterogeneity or in mean mobility, while the mobility for other plasma proteins is reduced in the typical way. Differences in acetylneuraminic acid content can thus be excluded as a probable cause of the heterogeneity. It seems more reasonable that the heterogeneity depends on formation of complexes between tL,-macroglobulin and other compounds since it has been shown that cr,-macroglobulin firmly links substances like trypsinz, somatotropin 4, plasmin and insulina*7. This work was supported No. 13X-581-01). Department of Clinical Chemistry, Malmb’ General Hospital, University of Lund (Sweden)
by the Swedish Medical Research
Council (Project
P. 0. C~ANROT C.-B. LAURELL
I M. SCHBNENBERGER,R.SCHMIDTBERGERANDH.E.SCHULTZE,Z. Naturforsch.,13 b(rg58)761. 2 C. B. LAURELL, Anal. Biochem., IO (1965) 358. 3 J. W. MEHL, W. O'CONNELL AND J. DEGROOT, Science, 145 (1964) 821. 4 D. R. HADDEN AND T. E. PROUT, Nature, 202 (1964) 1342. 5 H.E. SCHULTZE,N. HEIMBURGER, K. HEIDE, H.HAUPT, K. ST~RIKO AND G. SCHWICK,PYOC. 9th Europ. Sot. Haematol., Lisbon, 1963. Karger, Bawl/New York, 1963, p. 1315. 6 J. CLAUSEN, F. GJEDDE AND K. JORGENSEN,Proc. Sot. Exptl. Biol. Med., 112 (1963) 778. 7 G. R. ZAHND AND J. J. SCHEIDEGGER, Helv. Med. Acta, 30 (1963) 506.
Received Clin. Chim.
January zest, 1966 Acta,
14 (1966) 137-138