Dye-ligand affinity chromatography is one of the Affinity chromatography techniques used for protein purification of a complex mixture. Like general chromatography, but using dyes to apply on a support matrix of a column as the stationary phase that will allow a range of proteins with similar active sites to bind to, refers to as pseudo-affinity. [1] Synthetic dyes are used to mimic substrates or cofactors binding to the active sites of proteins which can be further enhanced to target more specific proteins. Follow with washing, the process of removing other non-target molecules, then eluting out target proteins out by changing pH or manipulate the salt concentration. The column can be reused many times due to the stability of immobilized dyes. It can carry out in a conventional way by using as a packed column, or in high-performance liquid chromatography (HPLC) column.[2]

Discovery

The serendipity discovery of dye-ligand ability is from a blue dye called blue dextran. The blue dye is used as a void volume (V0) marker for a gel filtration column. It has shown that the dye has a property to bind to some certain proteins like pyruvate kinase and elute out with the void volume. Later on, it was found that "cibacron blue FG3-A", reactive dye link to dextran, is responsible for the interaction with the proteins.[1][2]

Dye immobilization

The dyes are immobilized on the column matrix effectively, since usually the dyes link to a monochlorotriazine or dichlorotriazine ring (triazine dye). This type of dyes works especially well on a support matrix with hydroxyl group.[3] The commonly used supporting matrix would be cross-linked agarose (sepharose), sephadex, polyacrylamide, and silica.

An example for triazine linkage immobilization is Blue Sepharose, resulting from Cibacron blue FG3-A with monochlorotriazine covalently coupled with OH group of sepharose. This reaction form an ether linkage and also hydrogen chloride.[4]

C29H20ClN7O11S3 + C24H38O19 → C53H57N7O30S3 + HCl

Cibacron Blue FG3-A + Sepharose → Blue Sepharose + HCl

Reactive dyes

The dyes used in this type of chromatography are inexpensive and generally available as they are from textile industries called reactive dye. It contains chromophores that are often attached to a triazine ring. In textile industries, reactive dyes are used to dye material like cotton which is cellulose.

Commonly used reactive dyes for chromatography can be separated according to their color index name or functional group. Noted that each company has different trade names and slightly different formulas of the reactive dyes. Usually available commercially with sepharose as the supporting matrix in the form of packed columns.

Blue reactive dyes

Cibacron Blue F3GA

Cibacron Blue F3GA, Procion Blue HB, or Reactive blue 2 is a purinergic receptor antagonist, such as P2Y purinoceptor, and also an ATP receptor channels antagonist. It has a formula of C29H20ClN7O11S3 and a molecular weight of 774.2 g/mol.[5] Cibacron blue is soluble in water and DMSO, however insoluble in ethanol. In water, saturated concentration is reached at 12.92 mM with the help of sonication.[6] Cibacron Blue F3GA has a wide specificity for nucleotide-binding proteins or just a stereoselectivity electrostatic binding. It can be used to purify interferons, dehydrogenases, kinases, and serum albumin. For example, interferon purification from human gingival fibroblast extract using Cibacron Blue F3G-A on poly(2-hydroxyethyl methacrylate), the supporting matrix, in the form of cryogels. It has shown 97.6% purity of interferon. [7]

Blue MX-R

Blue MX-R or Reactive Blue 4 has a formula of C23H14Cl2N6O8S2 and a molecular weight of 637.4 g/mol.[8] It contains dichlorotriazine ring to the chromophore unlike Cibacron Blue F3GA. For a large scale protein purification, Blue MX-R can be used to purify protein such as lactate dehydrogenase (LDH).[2] In fast-protein liquid chromatography (FPLC) using Blue MX-R immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads, it was seen to separate lysozyme and bovine serum albumin (BSA), purified lysozyme from chicken albumin. [9]

Red reactive dyes

Red HE-3B

Red HE-3B or Reactive Red 120 has a formula of C44H30Cl2N14O20S6 and a molecular weight of 1338.1 g/mol, containing two monochlorotriazine rings. It is highly soluble in water.[10] The dehydrogenases binding ability of Red HE-3B is greater to NADP+ dependent dehydrogenases than NAD+ dependent dehydrogenases, vice versa for Cibacron Blue F3G-A. [11] It can be used to purify enterotoxins A, B, and C2 from Staphylococcus aureus using Procion Red HE-3B on sepharose, eluting out with 60 mM and 150 mM phosphate.[12]

Yellow reactive dyes

Yellow H-A

Yellow H-A or Reactive Yellow 3 has a formula of C21H17ClN8O7S2 and a molecular weight of 593 g/mol, containing a monochlorotriazine ring. On agarose as supporting matrix, it was seen to purify cholesteryl ester transfer protein.[13]

Brown reactive dyes

Brown MX-5BR

Brown MX-5BR or Reactive Brown 10 has a formula of C40H19Cl4CrN12Na2O12S2 and a molecular weight of 1163.6 g/mol, containing two dichlorotriazine rings. [14] Brown MX-5BR, for example, can be used to purify lysozyme, phosphinothricin acetyltransferase.[15] It also shown that it can elute tryptophanyl-tRNA synthetase using Trp as eluant, however, tryptophanyl-tRNA and tyrosyl-tRNA synthetase are the only t-RNA that can be elute out using Brown MX-5BR.[16]

References

  1. ^ a b McGettrick, Anne; Worrall, Margaret (2004). "Dye-Ligand Affinity Chromatography". Protein Purification Protocols. Methods in Molecular Biology. Vol. 244. Totowa, NJ: Humana Press. pp. 151–7. doi:10.1385/1-59259-655-x:151. ISBN 978-1-59259-655-3. PMID 14970553.
  2. ^ a b c Hsu, James; Boyer, Philip (1993). "Protein Purification by Dye-Ligand Chromatography" (PDF). Advances in Biochemical Engineering/Biotechnology. Springer. 49: 1–44. doi:10.1007/BFb0046571. ISBN 978-3-540-56440-9. PMID 8368124. Retrieved 14 February 2021.
  3. ^ Denizli, Adil; Pişkin, Erhan (30 October 2001). "Dye-ligand affinity systems". Journal of Biochemical and Biophysical Methods. 49 (1–3): 391–416. doi:10.1016/s0165-022x(01)00209-3. PMID 11694290.
  4. ^ Lower, Christopher R.; Pearson, James C. (1984). "Affinity chromatography on immobilized dyes". Part C: Enzyme Purification and Related Techniques. Methods in Enzymology. Vol. 104. pp. 97–113. doi:10.1016/S0076-6879(84)04085-4. ISBN 9780121820046. PMID 6144035.
  5. ^ "PubChem Compound Summary for CID 656725, Reactive Blue 2". Pub Chem. National Center for Biotechnology Information. Retrieved 12 March 2021.
  6. ^ "Procion Blue HB". MedChemExpress. Retrieved 12 March 2021.
  7. ^ Doğan, Ali; Özkara, Serpil; Sarı, Müfrettin; Uzun, Lokman; Denizli, Adil (2012). "Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system". Journal of Chromatography B. 893–894: 69–76. doi:10.1016/j.jchromb.2012.02.036. PMID 22445306.
  8. ^ "Reactive Blue 4". MedChemExpress. Retrieved 12 March 2021.
  9. ^ Wu, Feiyan; Zhu, Yan; Jia, Zhishen (2006). "Preparation of dye-ligand affinity chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their chromatographic properties". J Chromatogr A. 1134 (1–2): 45–50. doi:10.1016/j.chroma.2006.07.087. PMID 17034800.
  10. ^ "PubChem Compound Summary for CID 73264, Reactive Red 120". Pub Chem. National Center for Biotechnology Information.
  11. ^ Watson, D H; Harvey, M J; Dean, P D G (1978). "The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B". Biochemical Journal. 173 (2): 591–596. doi:10.1042/bj1730591. ISSN 0264-6021. PMC 1185813. PMID 29603.
  12. ^ Brehm, R D; Tranter, H S; Hambleton, P; Melling, J (1990). "Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography". Applied and Environmental Microbiology. 56 (4): 1067–72. doi:10.1128/aem.56.4.1067-1072.1990. PMC 184344. PMID 2339869.
  13. ^ "PubChem Compound Summary for CID 78686". Pub Chem. National Center for Biotechnology Information. Retrieved 14 March 2021.
  14. ^ "PubChem Compound Summary for CID 3081425, Procion Brown MX 5BR". Pub Chem. National Center for Biotechnology Information. Retrieved 14 March 2021.
  15. ^ Wang, C; Lee, T C; Crowley, K S; Bell, E (2013). "Purification of phosphinothricin acetyltransferase using Reactive brown 10 affinity in a single chromatography step". Protein Expression and Purification. 90 (2): 129–34. doi:10.1016/j.pep.2013.05.011. PMID 23748142.
  16. ^ Atkinson, T; Hammond, PM; HARTWELL, R D; HUGHES, P; SCAWEN, M D; SHERWOOD, R F; SMALL, D A; BRUTON, C J; HARVEY, M J; LOWE, C R (1981). "Triazine-dye affinity chromatography". Biochem Soc Trans. 9 (4): 290–3. doi:10.1042/bst0090290. PMID 7262447.
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