Trypsin chromatography

Figure 6. Sepharose 4B chromatography of trypsin-dextran conjugate (A). The sample contained approximately 145 mg dextran and 14 mg of trypsin. Chromatography of a mixture containing corresponding amounts of dextran and trypsin...

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Table 5.7 Theoretically predicted polypeptides from the trypsin digestion of S-lacto-globulin (/3LG) . Reprinted from J. Chromatogr., A, 763, Turula, V. E., Bishop, R. T., Ricker, R. D. and de Haseth, J. A., Complete structure elucidation of a globular protein by particle beam liquid chromatography-Fourier transform infrared spectrometry and electrospray liquid chromatography-mass spectrometry - Sequence and conformation of /3-lactoglobulin , 91-103, Copyright (1997), with permission from Elsevier Science...

Protein isolation with affinity precipitation has been discussed in detail by Mattiasson and co-workers (see, e.g. Galaev and Mattiassion, 1997) and they have provided an exhaustive tabulation. Polymers varied from alginate.s/chitosan to dextran to NIPAM. Examples of the used proteins are from trypsin, p-glucosidase, xylanase, alkaline protease, etc. It is remarkable that affinity precipitation can sometimes give results comparable to affinity chromatography. 

Titani, K., Sasagawa, T., Resing, K., and Walsh, K. A., A simple and rapid purification of commercial trypsin and chymostrypsin by reverse-phase high-performance liquid chromatography, Anal. Biochem., 123, 408, 1982. 

Fig. 9. Reversed-phase separations of cytochrome c digests obtained with trypsin-modified beads (left) and trypsin-modified monolithic reactor (right) in a tandem with a chromatographic column (Reprinted with permission from [90]. Copyright 1996 Wiley-VCH). Conditions digestion (left curve) trypsin-modified beads reactor, 50 mm x 8 mm i.d., 0.2 mg of cytochrome c, digestion buffer, flow rate 0.2 ml/min, 25 °C, residence time, 15 min (right curve) trypsin immobilized onto molded monolith other conditions the same as with trypsin-modified beads. Reversed-phase chromatography column, Nova-Pak C18,150 mm x 3.9 mm i.d., mobile phase gradient 0-70% acetonitrile in 0.1% aqueous trifluoroacetic acid in 15 min, flow rate, 1 ml/min, injection volume 20 pi, UV detection at 254 nm...

Figure 4.9 Anion-exchange liquid chromatography of acidic proteins. Column, Asahipak ES502N eluent, 20 min linear gradient of sodium chloride from 0 to 500 pM in 50 mM bis-tris/HCl buffer pH 7.0 flow rate, 1ml min-1 detection, UV 280 nm. Peaks-. 1, conalbumin (Mr 77000-88000, pi 6.0-6.8) 2, ovalbumin (Mt 45000, pi 4.6) 3, trypsin inhibitor (Mr 8000, pi 4.5) and 4, / -lactoglobulin (Mr 18400, pi 5.1).

Kasai, K., Ishii, S. Quantitative analysis of afSnity chromatography of trypsin. A new technique for investigation of protein-ligand interaction. J Biochem 1975, 77, 261-264. 

Proteins from albumin/immunoglobulin-depleted CSF (see Fig. 4) were digested with trypsin and the resultant peptides fractionated by two rounds of chromatography using cation exchange and reversed phase columns. 

Similarly, trypsin has been purihed from ground maize seed flour using STl-agarose, followed by cation exchange chromatography. 

A kininogen, which when incubated with trypsin or snake venom releases a material with a kinin-like ability to cause the contraction of smooth muscle, has been found in whey and concentrated by DEAE-cellulose chromatography (Leach et al. 1967). 

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