Serpins (Serine Protease Inhibitors)

Susannah J. Bauman1, Herbert C. Whinna1, Frank C. Church1

1 The University of North Carolina at Chapel Hill, Chapel Hill
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.7
DOI:  10.1002/0471140864.ps2107s26
Online Posting Date:  February, 2002
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Abstract

Serpins are a class of proteins involved in the regulation of serine and other types of proteases. In humans, the majority of serpins regulate the functions of proteases involved in the body's response to injury. This includes roles in coagulation, fibrinolysis, inflammation, wound healing, and tissue repair. Serpins have been implicated in various animal and human pathologies by the loss of a functional serpin gene through deletion or mutation, which results in a defect in functional protein. Examples of sestorically called antithrombin III) are first described. Then, protocols to determine the secondā€order rate constant of AT inhibition of thrombin in the absence and presence of heparin are presented. Also provided is a partial list of other serpins and their purification methods.

     
 
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Table of Contents

  • Basic Protocol 1: Purification of Human Antithrombin by Column Chromatography
  • Support Protocol 1: An Assay for Antithrombin in Eluted Fractions
  • Basic Protocol 2: An Assay of Antithrombin Inhibition in the Absence of Glycosaminoglycan—“Progressive Antithrombin Activity”
  • Basic Protocol 3: An Assay of Antithrombin Inhibition in the Presence of Glycosaminoglycan—“Heparin Cofactor Activity”
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of Human Antithrombin by Column Chromatography

  Materials
  • 12 to 14 U frozen citrated human plasma (available from the American Red Cross)
  • Soybean trypsin inhibitor
  • PEG 8000
  • 0.5 M (v/v) benzamidine solution (store up to 1 week at 4°C)
  • 1 M barium chloride (BaCl 2; store indefinitely at 4°C)
  • recipeTCE buffer (see recipe)
  • Heparin‐Sepharose in 5.0‐cm × 20‐cm column (heparin‐Sepharose can be purchased from Amersham Pharmacia Biotech or prepared using unbleached heparin and CnBr activation of Sepharose; see Griffith et al., )
  • recipeTCE buffer (see recipe) containing 2 M NaCl
  • 32°C water bath
  • 4‐liter beakers, clean
  • Magnetic stir plate and stir bars
  • 1‐liter centrifuge bottles
  • Centrifuge, 4°C
  • Fraction collector
  • Additional reagents and equipment for measuring protein concentration by spectrophotometry (unit 3.1), SDS‐PAGE (unit 10.1), and activity assay (see protocol 2)

Support Protocol 1: An Assay for Antithrombin in Eluted Fractions

  Materials
  • AT fractions (see protocol 1)
  • HNPN buffer (see recipe)
  • Thrombin/heparin solution (see recipe)
  • Chromozyme TH/polybrene solution (see recipe)
  • Microtiter plates coated with BSA (see recipe)
  • Kinetic plate reader (Molecular Devices)

Basic Protocol 2: An Assay of Antithrombin Inhibition in the Absence of Glycosaminoglycan—“Progressive Antithrombin Activity”

  Materials
  • recipeHNPN/polybrene solution (see recipe)
  • recipeChromozyme TH/polybrene solution (see recipe)
  • recipeAntithrombin (AT) solution (see recipe), purified and characterized (see protocol 1 and protocol 2)
  • recipeHNPN buffer (see recipe for buffer)
  • recipeThrombin solution (see recipe)
  • 50% (v/v) glacial acetic acid
  • recipeMicrotiter plates coated with BSA (see recipe)
  • Kinetic plate reader (Molecular Devices)

Basic Protocol 3: An Assay of Antithrombin Inhibition in the Presence of Glycosaminoglycan—“Heparin Cofactor Activity”

  Materials
  • recipeHeparin solutions (see recipe)
  • recipeAntithrombin (AT) solution (see recipe), purified and characterized (see protocol 1 and protocol 2)
  • recipeHNPN buffer (see recipe for buffer)
  • recipeThrombin solution (see recipe)
  • recipeChromozyme TH/polybrene solution (see recipe)
  • 50% (v/v) glacial acetic acid
  • recipeMicrotiter plates coated with BSA (see recipe)
  • Table‐top centrifuge suitable for microtiter plates
  • Kinetic plate reader (Molecular Devices)
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Figures

Videos

Literature Cited

Literature Cited
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   Church, F.C., Cunningham, D.D., Ginsburg, D., Hoffman, M., Stone, S.R., and Tollefsen, D.M. 1997. Chemistry and biology of serpins. Adv. Exp. Med. Biol. 425:358.
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   Lomas, D.A., Evans, D.L.I., Stone, S.R., Chang, W.‐S.W., and Carrell, R.W. 1993. Effect of the Z mutation on the physical and inhibitory properties of α1‐antitrypsin. Biochemistry 32:500‐508.
   Lottenberg, R., Hall, J.A., Blinder, M., Binder, E.P., and Jackson, C.M. 1983. The action of thrombin on peptide p‐nitroanilide substrates. Substrate selectivity and examination of hydrolysis under different reaction conditions. Biochim. Biophys. Acta. 742:539‐557.
   Mottonen, J., Strand, A., Symersky, J., Sweet, R.M., Danley, D.E., Geoghegan, K.F., Gerard, R.D., and Goldsmith, E.J. 1992. Structural basis of latency in plasminogen activator inhibitor‐1. Nature 355:270‐273.
   Nash, P., Whitty, A., Handwerker, J., Macen, J., and McFadden, G. 1998. Inhibitory specificity of the anti‐inflammatory myxoma virus serpin, SERP‐ 1. J. Biol. Chem. 273:20982‐20991.
   O'Reilly, M.S., Pirie‐Shepherd, S., Lane, W.S., and Folkman, J. 1999. Antiangiogenic activity of the cleaved conformation of the serpin antithrombin. Science 285:1926‐1928.
   Potempa, J., Korzus, E., and Travis, J.T. 1994. The serpin superfamily of proteinase inhibitors: Structure, function, and regulation. J. Biol. Chem. 269:15957‐15960.
   Pratt, C.W., Macik, B.G., and Church, F.C. 1989. Protein C inhibitor: Purification and proteinase reactivity. Thromb. Res. 53:595‐602.
   Quan, L.T., Caputo, A., Bleakley, R.C., Pickup, D.J., and Salvesen, G.S. 1995. Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A. J. Biol. Chem. 270:10377‐10379.
   Ray, C.A., Black, R.A., Kronheim, S.R., Greenstreet, T.A., Sleath, P.R., Salvesen, G.S., and Pickup, D.J. 1992. Viral inhibition of inflammation: Cowpox virus encodes an inhibitor of the interleukin‐1β converting enzyme. Cell 69:597‐604.
   Remold‐O'Donnell, E. 1993. The ovalbumin family of serpin proteins. FEBS Lett. 315:105‐108.
   Rosenberg, R.D. and Damus, P.S. 1973. The purification and mechanism of action of human antithrombin‐heparin cofactor. J. Biol. Chem. 248:6490‐6505.
   Schechter, I. and Berger, A. 1967. On the size of the active site in proteases I. Papain. Biochem. Biophys. Res. Commun. 27:157‐162.
   Skinner, R., Chang, W.S., Jin, L., Pei, X., Huntington, J.A., Abrahams, J.P., Carrell, R.W., and Lomas, D.A. 1998. Implications for function and therapy of a 2.9 A structure of binary‐complexed antithrombin. J. Mol. Biol. 283:9‐14.
   Stein, P.E. and Carrell, R.W. 1995. What do dysfunctional serpins tell us about molecular mobility and disease? Struct. Biol. 2:96‐113.
   Stein, P.E., Leslie, A.G.W., Finch, J.T., Turnell, W.G., Mclaughlin, P.J., and Carrell, R.W. 1990. Crystal structure of ovalbumin as a model for the reactive centre of serpins. Nature 347:99‐102.
   Suzuki, K., Nishioka, J., and Hashimoto, S. 1983. Protein C inhibitor: Purification from human plasma and characterization. J. Biol. Chem. 258:163‐168.
   Tollefsen, D.M. and Blank, M.K. 1981. Detection of a new heparin‐dependent inhibitor of thrombin in human plasma. J. Clin. Invest. 68:589‐596.
   Tollefsen, D.M., Majerus, D.W., and Blank, M.K. 1982. Heparin cofactor II: Purification and properties of a heparin‐dependent inhibitor of thrombin in human plasma. J. Biol. Chem. 257:2162‐2169.
   VanNostrand, W.E., Wagner, S.L., and Cunningham, D.D. 1988. Purification of a form of protease nexin 1 that binds heparin with a low affinity. Biochemistry 27:2172‐2177.
   Whisstock, J.C., Irving, J.A., Bottomley, S.P., Pike, R.N., and Lesk, A.M. 1999. Serpins in the Caenorhabditis elegans genome. Proteins 36:31‐41.
   Wun, T.‐C. and Reich, E. 1987. An inhibitor of plasminogen activation from human placenta: Purification and characterization. J. Biol. Chem. 262:3646‐3653.
Key References
   Church et al., 1997.
  Proceedings of the International Symposium on the chemistry and biology of serpins held April 13 to 16, 1996, in Chapel Hill, North Carolina. Provides overviews of numerous serpins.
   Gettins and Olson, 1996.
  Provides basic descriptions of action of various serpins.
   Griffith et al., 1985a.
  Describes the purification and subsequent assay of human antithrombin III and heparin cofactor II.
   Silverman, G.A., Bird, P.I., Carrell, R.W., Church, F.C., Coughlin, P.B., Gettins, P.G., Irving, J.A., Lomas, D.A., Luke, C.J., Moyer, R.W., Pemberton, P.A., Remold‐O'Donnell, E., Salvesen, G.S., Travis, J., and Whisstock, J.C. 2001. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J. Biol. Chem. 276:33293‐33296.
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