title: Residual transglutaminase in collagen - effects, detection, quantification, and removal.
abstract: In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) for microbial transglutaminase (mTG) from Streptomyces mobaraensis to overcome the lack of a quantification method for mTG. We further performed a detailed follow-on-analysis of insoluble porcine collagen type I enzymatically modified with mTG primarily focusing on residuals of mTG. Repeated washing (4 ×) reduced mTG-levels in the washing fluids but did not quantitatively remove mTG from the material (p < 0.000001). Substantial amounts of up to 40% of the enzyme utilized in the crosslinking mixture remained associated with the modified collagen. Binding was non-covalent as could be demonstrated by Western blot analysis. Acidic and alkaline dialysis of mTG treated collagen material enabled complete removal the enzyme. Treatment with guanidinium chloride, urea, or sodium chloride was less effective in reducing the mTG content.
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University, Munich, Germany. winfried.schloegl@gmx.de
authors.forename: W
authors.initials: W
authors.lastname: Schloegl
authors.forename: A
authors.initials: A
authors.lastname: Klein
authors.forename: R
authors.initials: R
authors.lastname: Fürst
authors.forename: U
authors.initials: U
authors.lastname: Leicht
authors.forename: E
authors.initials: E
authors.lastname: Volkmer
authors.forename: M
authors.initials: M
authors.lastname: Schieker
authors.forename: S
authors.initials: S
authors.lastname: Jus
authors.forename: G M
authors.initials: GM
authors.lastname: Guebitz
authors.forename: I
authors.initials: I
authors.lastname: Stachel
authors.forename: M
authors.initials: M
authors.lastname: Meyer
authors.forename: M
authors.initials: M
authors.lastname: Wiggenhorn
authors.forename: W
authors.initials: W
authors.lastname: Friess
mesh_terms: D000818:Animals; D015153:Blotting, Western; D024042:Collagen Type I / Q000378:metabolism*; D003956:Dialysis / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D012965:Sodium Chloride / Q000737:chemistry; D013302:Streptomyces / Q000201:enzymology; D013552:Swine; D011503:Transglutaminases / Q000378:metabolism*; D014508:Urea / Q000737:chemistry
chemical_list: D024042:Collagen Type I; D012965:Sodium Chloride; D014508:Urea; D011503:Transglutaminases; D019791:Guanidine