Tweezing-adsorptive bubble separation. Analytical method for the selective and high enrichment of metalloenzymes.
A novelly developed tweezing-adsorptive bubble separation (ABS) method for the enrichment of metalloenzymes (laccase C and horseradish peroxidase) is introduced. The method is based on the chelation of the enzymes' active center and can also be applied for analysis. N-(2-acetamido)iminodiacetic acid served as a chelator and was synthesized with an octyl unit to become ADA-C8. Laccase was enriched 13.3-fold (66.31% recovery) and HPOX 17.8-fold (85.34%) without a significant loss of enzymatic activity. To prove that the entire enzyme is tweezed at the active center, ABS trials were done using ADA-C8 already complexed with Cu2+ and Fe3+. As only marginal enrichment occurred (ER laccase, 0.17; ER HPOX, 0.44), no chelating effect was concluded. It was determined how the chelation toward the active center was directed by applying other chelators such as EDTA, NTA, N,N-dimethylaminoglycine, oxalic acid, malonic acid, adipinic acid, and tripropylamine, which are similar in structure to ADA-C8. The results concluded that the chelation is 3-fold coordinated on the type 1 copper center of laccase, whereas that of HPOX only 1-fold at Fe3+ and additionally at the cationic amino acid arginine, which is also located at the active center. Tweezing-ABS has been proven to selectively and effectively enrich metalloenzymes.
Gerken
Birte M
BM
Department of Chemical-Technical Analysis and Chemical Food Technology, Research Center Weihenstephan for Brewing and Food Quality, Technical University of Munich, Weihenstephaner Steig 23, D-85354 Freising-Weihenstephan, Germany.
Wattenbach
Carsten
C
Linke
Diana
D
Zorn
Holger
H
Berger
Ralf G
RG
Parlar
Harun
H
D000042:Absorption; D001665:Binding Sites; D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D042845:Laccase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008667:Metalloproteins / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D017434:Protein Structure, Tertiary
D002614:Chelating Agents; D008667:Metalloproteins; D042845:Laccase; D006735:Horseradish Peroxidase