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. 1986 Apr 15;235(2):559–567. doi: 10.1042/bj2350559

Purification and characterization of human alpha 2-macroglobulin conformational variants by non-ideal high performance size-exclusion chromatography.

S L Gonias, P A Roche, S V Pizzo
PMCID: PMC1146721  PMID: 2427074

Abstract

Human alpha 2-macroglobulin (alpha 2M) was eluted as a single nondispersed peak from a TSK-G4000SW size exclusion chromatography column equilibrated in 20 mM-sodium phosphate/100 mM-NaCl, pH 7.2 (PBS). The void volume and total accessible volume of the column were 6.08 ml and 14.42 ml. The elution volume (Ve) of native alpha 2M was 9.20 +/- 0.04 ml. The Ve was altered minimally by changing the ionic strength or adding ethanol to the equilibration buffer. Ternary alpha 2M-trypsin, containing 2 mol of proteinase/mol of inhibitor, and alpha 2M-methylamine failed to be eluted in well-defined peaks when the column was equilibrated in PBS. The majority of either preparation was recovered slowly at Ve values greater than 14.5 ml, reflecting significant nonideal interactions with the support structure. Addition of 10% ethanol or increased ionic strength in the equilibration buffer independently caused either form of reacted alpha 2M to be eluted in a distinct peak at decreased Ve, suggesting that the nonideal interactions included hydrophobic and electrostatic adsorption. When the equilibration buffer was 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2, partial resolution of ternary alpha 2M-trypsin and alpha 2M-methylamine was obtained with a single column run. The Ve values of ternary alpha 2M-trypsin and alpha 2M-methylamine in this buffer were 13.15 +/- 0.08 ml and 11.94 +/- 0.14 ml, respectively. The Ve of native alpha 2M was 8.84 +/- 0.03 ml. The resolving capacity of TSK-G4000SW was exploited to purify native alpha 2M rapidly and efficiently from solutions that contained significant amounts of either ternary alpha 2M-trypsin or binary alpha 2M-trypsin (1 mol of proteinase/mol of inhibitor). This purification was complete within the limits of sensitivity of denaturing and nondenaturing polyacrylamide-gel electrophoresis. alpha 2M-plasmin was well resolved from native alpha 2M. The Ve of alpha 2M-plasmin was 12.88 +/- 0.32 ml in 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2. A number of procedures were used to prepare solutions with up to 90% binary alpha 2M-trypsin. The Ve of binary alpha 2M-trypsin in these various solutions was intermediate between the values of native alpha 2M and ternary alpha 2M-trypsin. The conformations of binary and ternary complex, as reflected by mobility in nondenaturing electrophoresis, were identical, confirming previous results. Finally, in the binary alpha 2M-trypsin complex, the single trypsin cleaved more than two, and as many as all four alpha 2M subunits.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barrett A. J., Brown M. A., Sayers C. A. The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule. Biochem J. 1979 Aug 1;181(2):401–418. doi: 10.1042/bj1810401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrett A. J., Starkey P. M. The interaction of alpha 2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem J. 1973 Aug;133(4):709–724. doi: 10.1042/bj1330709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bieth J. G., Tourbez-Perrin M., Pochon F. Inhibition of alpha 2-macroglobulin-bound trypsin by soybean trypsin inhibitor. J Biol Chem. 1981 Aug 10;256(15):7954–7957. [PubMed] [Google Scholar]
  4. Björk I., Fish W. W. Evidence for similar conformational changes in alpha 2-macroglobulin on reaction with primary amines or proteolytic enzymes. Biochem J. 1982 Nov 1;207(2):347–356. doi: 10.1042/bj2070347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brockway W. J., Castellino F. J. Measurement of the binding of antifibrinolytic amino acids to various plasminogens. Arch Biochem Biophys. 1972 Jul;151(1):194–199. doi: 10.1016/0003-9861(72)90488-2. [DOI] [PubMed] [Google Scholar]
  6. Castellino F. J., Sodetz J. M., Brockway W. J., Siefring G. E., Jr Streptokinase. Methods Enzymol. 1976;45:244–257. doi: 10.1016/s0076-6879(76)45024-3. [DOI] [PubMed] [Google Scholar]
  7. Chase T., Jr, Shaw E. p-Nitrophenyl-p'-guanidinobenzoate HCl: a new active site titrant for trypsin. Biochem Biophys Res Commun. 1967 Nov 30;29(4):508–514. doi: 10.1016/0006-291x(67)90513-x. [DOI] [PubMed] [Google Scholar]
  8. Cummings H. S., Castellino F. J. Interaction of human plasmin with human alpha 2-macroglobulin. Biochemistry. 1984 Jan 3;23(1):105–111. doi: 10.1021/bi00296a017. [DOI] [PubMed] [Google Scholar]
  9. Cummings H. S., Pizzo S. V., Strickland D. K., Castellino F. J. Effect of methylamine and plasmin on the conformation of human alpha 2-macroglobulin as revealed by differential scanning calorimetric analysis. Biophys J. 1984 Apr;45(4):721–724. doi: 10.1016/S0006-3495(84)84214-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. David G. S., Reisfeld R. A. Protein iodination with solid state lactoperoxidase. Biochemistry. 1974 Feb 26;13(5):1014–1021. doi: 10.1021/bi00702a028. [DOI] [PubMed] [Google Scholar]
  11. Deutsch D. G., Mertz E. T. Plasminogen: purification from human plasma by affinity chromatography. Science. 1970 Dec 4;170(3962):1095–1096. doi: 10.1126/science.170.3962.1095. [DOI] [PubMed] [Google Scholar]
  12. Eccleston E. D., Howard J. B. Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation. J Biol Chem. 1985 Aug 25;260(18):10169–10176. [PubMed] [Google Scholar]
  13. Feldman S. R., Gonias S. L., Ney K. A., Pratt C. W., Pizzo S. V. Identification of "embryonin" as bovine alpha 2-macroglobulin. J Biol Chem. 1984 Apr 10;259(7):4458–4462. [PubMed] [Google Scholar]
  14. Feldman S. R., Gonias S. L., Pizzo S. V. Model of alpha 2-macroglobulin structure and function. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5700–5704. doi: 10.1073/pnas.82.17.5700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ganrot P. O. Determination of alpha-2-macroglobulin as trypsin-protein esterase. Clin Chim Acta. 1966 Oct;14(4):493–501. doi: 10.1016/0009-8981(66)90037-4. [DOI] [PubMed] [Google Scholar]
  16. Ganrot P. O. Inhibition of plasmin activity by alpha-2-macroglobulin. Clin Chim Acta. 1967 May;16(2):328–329. doi: 10.1016/0009-8981(67)90201-x. [DOI] [PubMed] [Google Scholar]
  17. Gonias S. L., Einarsson M., Pizzo S. V. Catabolic pathways for streptokinase, plasmin, and streptokinase activator complex in mice. In vivo reaction of plasminogen activator with alpha 2-macroglobulin. J Clin Invest. 1982 Aug;70(2):412–423. doi: 10.1172/JCI110631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gonias S. L., Feldman S. R., Pratt C. W., Pizzo S. V. Conformation-specific precipitation of human alpha 2-macroglobulin by divalent zinc or calf thymus histone H3. Arch Biochem Biophys. 1984 Sep;233(2):462–468. doi: 10.1016/0003-9861(84)90468-5. [DOI] [PubMed] [Google Scholar]
  19. Gonias S. L., Pizzo S. V. Characterization of functional human alpha 2-macroglobulin half-molecules isolated by limited reduction with dithiothreitol. Biochemistry. 1983 Feb 1;22(3):536–546. doi: 10.1021/bi00272a003. [DOI] [PubMed] [Google Scholar]
  20. Gonias S. L., Pizzo S. V. Chemical and structural modifications of alpha 2-macroglobulin: effects on receptor binding and endocytosis studied in an in vivo model. Ann N Y Acad Sci. 1983;421:457–471. doi: 10.1111/j.1749-6632.1983.tb18139.x. [DOI] [PubMed] [Google Scholar]
  21. Gonias S. L., Pizzo S. V. Conformation and protease binding activity of binary and ternary human alpha 2-macroglobulin-protease complexes. J Biol Chem. 1983 Dec 10;258(23):14682–14685. [PubMed] [Google Scholar]
  22. Gonias S. L., Pizzo S. V. Reaction of human alpha 2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure. Biochemistry. 1983 Oct 11;22(21):4933–4940. doi: 10.1021/bi00290a009. [DOI] [PubMed] [Google Scholar]
  23. Gonias S. L., Reynolds J. A., Pizzo S. V. Physical properties of human alpha 2-macroglobulin following reaction with methylamine and trypsin. Biochim Biophys Acta. 1982 Aug 10;705(3):306–314. doi: 10.1016/0167-4838(82)90252-7. [DOI] [PubMed] [Google Scholar]
  24. Hall P. K., Roberts R. C. Physical and chemical properties of human plasma alpha2-macroglobulin. Biochem J. 1978 Jul 1;173(1):27–38. doi: 10.1042/bj1730027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Harpel P. C., Mosesson M. W. Degradation of human fibrinogen by plasms alpha2-macroglobulin-enzyme complexes. J Clin Invest. 1973 Sep;52(9):2175–2184. doi: 10.1172/JCI107402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Harpel P. C. Studies on human plasma alpha 2-macroglobulin-enzyme interactions. Evidence for proteolytic modification of the subunit chain structure. J Exp Med. 1973 Sep 1;138(3):508–521. doi: 10.1084/jem.138.3.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hefti F. High-performance size-exclusion chromatography: a buffer for the reliable determination of molecular weights of proteins. Anal Biochem. 1982 Apr;121(2):378–381. doi: 10.1016/0003-2697(82)90496-1. [DOI] [PubMed] [Google Scholar]
  28. Howell J. B., Beck T., Bates B., Hunter M. J. Interaction of alpha 2-macroglobulin with trypsin, chymotrypsin, plasmin, and papain. Arch Biochem Biophys. 1983 Feb 15;221(1):261–270. doi: 10.1016/0003-9861(83)90143-1. [DOI] [PubMed] [Google Scholar]
  29. Imber M. J., Pizzo S. V. Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin. J Biol Chem. 1981 Aug 10;256(15):8134–8139. [PubMed] [Google Scholar]
  30. Kopaciewicz W., Regnier F. E. Nonideal size-exclusion chromatography of proteins: effects of pH at low ionic strength. Anal Biochem. 1982 Oct;126(1):8–16. doi: 10.1016/0003-2697(82)90102-6. [DOI] [PubMed] [Google Scholar]
  31. Kurecki T., Kress L. F., Laskowski M., Sr Purification of human plasma alpha 2 macroglobulin and alpha 1 proteinase inhibitor using zinc chelate chromatography. Anal Biochem. 1979 Nov 1;99(2):415–420. doi: 10.1016/s0003-2697(79)80026-3. [DOI] [PubMed] [Google Scholar]
  32. McLellan T. Electrophoresis buffers for polyacrylamide gels at various pH. Anal Biochem. 1982 Oct;126(1):94–99. doi: 10.1016/0003-2697(82)90113-0. [DOI] [PubMed] [Google Scholar]
  33. Neville D. M., Jr Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J Biol Chem. 1971 Oct 25;246(20):6328–6334. [PubMed] [Google Scholar]
  34. Pochon F., Bieth J. G. Separation of free and chymotrypsin-bound alpha 2-macroglobulin by affinity chromatography. Its use to demonstrate that the two chymotrypsin-binding sites of alpha 2-macroglobulin are equivalent and independent. J Biol Chem. 1982 Jun 25;257(12):6683–6685. [PubMed] [Google Scholar]
  35. Regnier F. E., Gooding K. M. High-performance liquid chromatography of proteins. Anal Biochem. 1980 Mar 15;103(1):1–25. doi: 10.1007/978-1-4615-6728-8_1. [DOI] [PubMed] [Google Scholar]
  36. Salvesen G. S., Barrett A. J. Covalent binding of proteinases in their reaction with alpha 2-macroglobulin. Biochem J. 1980 Jun 1;187(3):695–701. doi: 10.1042/bj1870695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Salvesen G. S., Sayers C. A., Barrett A. J. Further characterization of the covalent linking reaction of alpha 2-macroglobulin. Biochem J. 1981 May 1;195(2):453–461. doi: 10.1042/bj1950453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Saunders R., Dyce B. J., Vannier W. E., Haverback B. J. The separation of alpha-2 macroglobulin into five components with differing electrophoretic and enzyme-binding properties. J Clin Invest. 1971 Nov;50(11):2376–2383. doi: 10.1172/JCI106736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sottrup-Jensen L., Petersen T. E., Magnusson S. A thiol-ester in alpha 2-macroglobulin cleaved during proteinase complex formation. FEBS Lett. 1980 Dec 1;121(2):275–279. doi: 10.1016/0014-5793(80)80361-9. [DOI] [PubMed] [Google Scholar]
  40. Sottrup-Jensen L., Stepanik T. M., Kristensen T., Wierzbicki D. M., Jones C. M., Lønblad P. B., Magnusson S., Petersen T. E. Primary structure of human alpha 2-macroglobulin. V. The complete structure. J Biol Chem. 1984 Jul 10;259(13):8318–8327. [PubMed] [Google Scholar]
  41. Straight D. L., McKee P. A. Effect of protease binding by alpha 2-macroglobulin on intrinsic fluorescence. Biochemistry. 1982 Sep 14;21(19):4550–4556. doi: 10.1021/bi00262a006. [DOI] [PubMed] [Google Scholar]
  42. Strickland D. K., Bhattacharya P., Olson S. T. Kinetics of the conformational alterations associated with nucleophilic modification of alpha 2-macroglobulin. Biochemistry. 1984 Jul 3;23(14):3115–3124. doi: 10.1021/bi00309a002. [DOI] [PubMed] [Google Scholar]
  43. Swenson R. P., Howard J. B. Structural characterization of human alpha2-macroglobulin subunits. J Biol Chem. 1979 Jun 10;254(11):4452–4456. [PubMed] [Google Scholar]
  44. Van Leuven F., Cassiman J. J., Van den Berghe H. Functional modifications of alpha 2-macroglobulin by primary amines. I. Characterization of alpha 2 M after derivatization by methylamine and by factor XIII. J Biol Chem. 1981 Sep 10;256(17):9016–9022. [PubMed] [Google Scholar]
  45. Wang D., Wu K., Feinman R. D. The reaction of alpha 2-macroglobulin-bound trypsin with soybean trypsin inhibitor. J Biol Chem. 1981 Nov 10;256(21):10934–10940. [PubMed] [Google Scholar]

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