title: Purification and biochemical characterization of an organic-solvent-tolerant thioredoxin from dromedary pancreas.
abstract: We purified to homogeneity and characterized a heat stable thioredoxin which catalyzes thiol/disulfide exchange reaction, for the first time from dromedary pancreas. The purification involved heat and acidic treatment (90 °C; pH 2.5), precipitation by ammonium sulphate and ethanol, respectively followed by sequential column chromatography reverse HPLC column, and it resulted in an apparently pure protein after a 217-fold purification with a final yield of 55% of the initial thioredoxin activity. The thioredoxin preparation obtained was homogeneous as judged by polyacrylamide gel electrophoresis and the presence of valine as the only NHt-terminal amino acid. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,302.9 Da. The first 40 amino-acid residues at the N-terminal extremity of purified DrTrx was determined by automatic Edman degradation and showed a high sequence homology with known Thioredoxin. It contained he tetrapeptide-Cys-Gly-Pro-Cys-, which constitutes the active site of mammalian thioredoxins. DrTrx activity was compatible with the presence of organic solvents and the maximum activity appeared at pH 7.5 using the insulin precipitation assay. Thioredoxin stability in the presence of organic solvents, as well as in acidic and alkaline pHs and at high temperatures makes it a good candidate for its application in pharmaceutical and food industry.
authors.affiliation: Biochemistry Department, Science College, King Saud University, P.O. box 22452, Riyadh, 11495, Saudi Arabia. abirghanouchi@yahoo.fr
authors.forename: Abir G
authors.initials: AG
authors.lastname: Ben Bacha
authors.forename: Hafedh
authors.initials: H
authors.lastname: Mejdoub
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002162:Camelus / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000737:chemistry* / Q000378:metabolism; D055550:Protein Stability; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D013879:Thioredoxins
references.citation: J Biol Chem. 1974 Jan 10;249(1):205-10
references.pmid: 4809626
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: DNA Seq. 1994;4(4):277-9
references.pmid: 7987015
references.citation: Curr Opin Pharmacol. 2007 Aug;7(4):392-7
references.pmid: 17611157
references.citation: Annu Rev Biochem. 1985;54:237-71
references.pmid: 3896121
references.citation: Proc Natl Acad Sci U S A. 1975 Jun;72(6):2305-9
references.pmid: 1094461
references.citation: Biochim Biophys Acta. 2008 Nov;1780(11):1368-82
references.pmid: 18206121
references.citation: EMBO J. 1984 Jul;3(7):1443-9
references.pmid: 6378624
references.citation: Comp Biochem Physiol B Biochem Mol Biol. 2008 Apr;149(4):630-9
references.pmid: 18255328
references.citation: Eur J Biochem. 2000 Oct;267(20):6102-9
references.pmid: 11012661
references.citation: J Agric Food Chem. 2008 Aug 27;56(16):7146-50
references.pmid: 18636684
references.citation: Eur J Biochem. 1972 Mar 15;26(1):119-24
references.pmid: 5065076
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Proteins. 1991;11(1):13-28
references.pmid: 1961698
references.citation: Ital J Biochem. 2003 Mar;52(1):28-32
references.pmid: 12833635
references.citation: IUBMB Life. 2001 Jul;52(1-2):3-6
references.pmid: 11795590
references.citation: J Biol Chem. 1981 Aug 10;256(15):7990-7
references.pmid: 7263636
references.citation: Annu Rev Immunol. 1997;15:351-69
references.pmid: 9143692
references.citation: J Biol Chem. 1973 Feb 25;248(4):1219-23
references.pmid: 4405641
references.citation: Genome Biol. 2009;10(4):R42
references.pmid: 19393038
references.citation: Dev Cell. 2002 Mar;2(3):251-2
references.pmid: 11879627
references.citation: J Physiol. 2001 Feb 15;531(Pt 1):1-11
references.pmid: 11179387
references.citation: FASEB J. 2003 Nov;17(14):2088-90
references.pmid: 12958160
references.citation: Structure. 1994 Jun 15;2(6):503-22
references.pmid: 7922028
references.citation: J Biol Chem. 1964 Oct;239:3436-44
references.pmid: 14245400
references.citation: J Biol Chem. 1979 Oct 10;254(19):9627-32
references.pmid: 385588
references.citation: J Allergy Clin Immunol. 2006 Mar;117(3):676-81
references.pmid: 16522470
references.citation: J Biol Chem. 1994 Nov 25;269(47):29382-4
references.pmid: 7961915
references.citation: J Mol Biol. 1990 Mar 5;212(1):167-84
references.pmid: 2181145
references.citation: J Biol Chem. 1988 Apr 5;263(10):4984-90
references.pmid: 2832416
references.citation: Structure. 1994 Sep 15;2(9):853-68
references.pmid: 7812718
references.citation: Mamm Genome. 2007 Mar;18(3):197-209
references.pmid: 17406940
references.citation: Biochemistry. 1993 Sep 21;32(37):9701-8
references.pmid: 8373774
references.citation: Anal Biochem. 1985 Sep;149(2):516-20
references.pmid: 4073506