Thrombin Purification in the second half of the 20th century
               Thrombin was “discovered” as fibrin ferment in the 19th century.  Debate as to whether thrombin was an enzyme continued as late as the 1940s. Work from various laboratories in the 1950s showed that thrombin was an enzyme based on the release of what are known today as fibrinopeptides.  The lack of progress on the understanding of thrombin and many other proteins was limited by the lack of purified materials. The following is a discussion of the road to purified thrombin during the period of 1954 through 1980.  This, for me, an entertaining story both for what was seen and what was missed.   I should note that I am currently working on a book on thrombin which should be available by the middle of 2020.   The following is taken, in part, from that work.
There was a marked increase between 1950 and 1960 in the various technologies available for the study of proteins. This activity was driven by three related factors; First, there was an efflux of talented scientists from WWII service.  Second, various technologies developed during WWII became available for use in the academic and commercial sectors (1).  Third, the US government (and other world governments) decided that the support of basic science research was a good investment (2).
.   Ion-exchange resins date to the 19th century (3) and were put a solid footing in 1935 (4).  Ion-exchange resins were further developed during WWII for the of water for desalinization and for the recovery of metal ions from manufacturing processes (5,6).   Subsequently it was shown that these resins could be used for the separation of peptides and proteins (7).   A technique for the determination of amino acids by ion-exchange chromatography was also developed during this time frame (8,9) replacing tedious microbiological and colorimetric methods.  The Edman degradation was developed for the determination the sequence of amino acids in peptides and proteins (10,11).  Similar advances were made in spectroscopy, electrophoresis, and ultracentrifugation.   These various technologies permitted advances in all areas of biochemistry including the purification and characterization of proteins.
P Stroier Rasmussen at the University of Copenhagen took advantage of the availability of ion-exchange resins to purify thrombin using Amberlite IRC-50 (12).  Rasmussen observed that despite an isoelectric point between 4.7(low) and 6(high), thrombin behaved as a cation at pH 7.0 in sodium phosphate buffer.  His choice of IRC-50 (cation exchange sin) was based on previous observations that thrombin bound to glass (13,14) which bears a diffuse negative charge (15).   Rasmussen did suggest that regardless of iselectric point, thrombin may not be an acidic protein (isoelectric point, Ip < 7.0).  As discussed below, these results were rationalized by the elucidation of the presence of basic exodomains.  Miller subsequently showed that impurities in either biothrombin [thrombin prepared by activation of prothrombin with biological factors(tissue factor, factors VII, X, V, calcium ions)  or citrate thrombin (prothrombin activated In 25% trisodium citrate) could be removed by precipitation with Rivanol™ (ethacridine lactate; 7-ethoxyacridine-3,9-diamine; 2-hydroxypropanoic acid) but found that a more efficient procedure used adsorption and subsequent elution of crude thrombin from CG-50 resin previously equilibrated with ethacridine lactate (16).  The ion-exchange resin used by Miller is similar to that used by Rasmussen; both IRC-50 and CG-50 are weak cation exchange resins containing a carboxyl group.  It is my sense that Amberlite IRC-50 ion-exchange resin is no longer available.    Ethacridine lactate has been used in the purification of antibody fragments produced in Escherichia coli where it precipitates host cell protein (17) while In earlier work, separation of by precipitation of free ligand (thyroxin)  from bound ligand in a radioimmunoassay was accomplished with ethacridine lactate. (18).   Miller (19) extended his work on thrombin purification with the chromatography of biothrombin on CG-50 resin in either cacodylate buffer tris buffer at pH 7.0.  John Fenton working also the New York State Department of Health improved the Miller process for the purification of human thrombin on CG-50 starting with human Cohn III paste (20,21).  Prothrombin was purified from Cohn III paste by barium citrate precipitation and elution and activated with thromboplastin. The crude thrombin was purified by chromatography on CG-50 at pH 8.0.  It is fair to say the provision of this purified thrombin by John Fenton enabled great amount of work on thrombin by a number of other investigators for the next several decades.   It is my sense that Dr. Fenton never received full credit for his contributions to the study of thrombin.  At the same I developed a procedure for the purifi2ation of bovine thrombin using Sulfoethyl-Sephadex (22).  I should note that we needed the thrombin for our work on factor VIII (23) but thrombin became of major interest to my laboratory for the next decade.   My choice of Sulfoethyl-Sephadex was based on a conversation with Stanford Moore when I was at the Rockefeller University who mentioned they had substituted sulfoethyl-Sephadex for IRC-50 in work on pancreatic ribonuclease.   The method was later adapted to Sulfopropyl-Sephadex and was shown to be effective for human thrombin (24).  Neither John Fenton nor I picked up on the Rasmussen comment about thrombin as an acidic protein in our studies on thrombin purification.  However, Fenton and others shortly later developed the concept of the exosite concept for thrombin (25,26) which provides a rationale for chromatographic behavior of thrombin on cation-exchange matrices. In going back and looking at the Rasmussen paper, I realize that I ignored the last part of the paper which discussed the question of the concept of thrombin as an acidic protein; my bad!  I have learned to be more careful in reading papers.
1.   Miller, C.R., Learning from of history. World War II and the cultures of high technology, J.Bus.Tech.Commun. 12, 288-315  1988.
2.  Waterman, A.J., The National Science Foundation. Its organization and purpose, Am.J.Physics 20, 73-77, 1952.
3.   Way, J.T., Power of soil to absorb manure, J.Roy.Agr.Soc.Engl. 11:313, 1850 (cited in Colella, C., Ion exchange equilibria in zeolite minerals, Mineralium Deposita 31, 554-562, 1996)
4. Adams, B.A. and Holmes, E.I., Absorptive properties of synthetic zeolites, J.Soc.Chem.Ind. 54,1T-6T, 1935.
5.  Tompkins, E.F., Ion exchange separations, Anal.Chem. 22, 1352-1359, 1950
6.  Nachod, F.C., Ion exchange, The Scientific Monthly 70, 189-194, 1950.
7 . Moore, S. and Stein, W.H., Column chromatography of peptides and proteins, Adv.Prot.Chem. 11, 1011-1236, 1956.
8.  Spackman, D.H., Stein, W.H., and Moore, S., Automatic recording apparatus for use in the chromatography of amino acids, Anal.Chem. 30, 1190-1206, 1958.
9.  Hirs, C.H.W., Moore, S., and Stein, W.H., The sequence of amino acid residues in performic acid-oxidized ribonuclease, J.Biol.Chem. 235, 633-647, 1960.
10.  Edman, P., A method for the determination of amino acid sequence in peptides, Arch.Biochem. 22, 475-476, 1949.
11.   Edman, P., Phenylthiohydantoins in protein analysis, Ann.N.Y.Acad.Sci. 88, 602-610, 1960.
12.  Rasmussen, P.S, Purification of thrombin by chromatography, Biochim.Biophys.Acta 16, 157-158, 1955.
13.   Waugh, D.F. and Livingstone, B.J., Kinetics of the interaction of bovine fibrinogen and thrombin, J.Phys.Chem. 55, 1206-1218, 1951.
14.  Waugh, D.F. Anthony, L.J. and Ng, N., The interaction of thrombin with borosilicate glass surfaces, J.Biomed.Mater.Res. 9, 511-536, 1975.
15  Behrens, S.H. and Grier, D.C., The charge of glass and silica surfaces, J.Chem.Phys. 115(14):6716, 2001.
16. Miller, K.D., Rivanol, resin and the isolation of thrombin, Nature 184, 450, 1959.
17.  Persson, J. and Lester, P., Purification of antibody and antibody-fragment from E.coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipitation agent, Biotechnol.Bioeng. 87, 424-434, 2004.
18. Bhupal, V. and Mani, R.S., Separation of bound and free ligand by ethacridine (Rivenol) in thyroxin radioimmunoassay, Clin.Chem. 29, 1937-1940, 1983.
19.  Miller, K.D. and Copeland, W.H., Human thrombin: Isolation and stability, Exptl.Mol.Pathol. 4, 431-437, 1965.
20.  Fenton, J.W., II., Campbell, W.P., Harrington, J.C., and Miller, K.D., Large scale preparation and preliminary characterization of human thrombin, Biochim.Biophys.Acta 229, 26-32, 1971.
21. Fenton, J.W.,2nd, Fasco, M.J., Stackrow, A.B., et al., Human thrombins. Production, evaluation, and properties of α-thrombin, J.Biol.Chem. 252, 3587-3598, 1977.
22. Lundblad, R .L, A rapid method for the purification of bovine thrombin and the reaction of the purified enzyme with phenylmethylsulfonyl fluoride, Biochemistry 10, 2501-2506, 1971.
23.  Vogel, C.N., Parfitt, H.E.,Jr., Kingdon, H.S., and Lundblad, R.L, Preparation of modified bovine factor VIII with enhanced biological activity using insoluble-trypsin columns, Thromb.Diath.Haemorrh. 30, 229-234, 1973.
24.  Lundblad, R.L., Kingdon, H.S., and Mann, K.G., Thrombin, Methods Enzymol. 45, 156-177, 1976.
25.  Fenton,  J.W.,2nd, Olson, T.A., Zabinksi,  M.P., and Wilner,G.D., Anion-binding exosite of human α-thrombin and fibrin(ogen) recognition, Biochemistry 27, 7106-7112, 1988.
26. Fenton, J.W., 2nd, Witting, J.I., Pouliott, C., and  Fareed, J., Thrombin anion-binding exosite interactions with heparin and various polyanions, Ann.N.Y.Acad.Sci. 556, 158-165, 1989.
© Roger L Lundblad, Chapel Hill, North Carolina, USA June 25, 2019