Application of zero-length cross-linking to form lysozyme, horseradish peroxidase and lysozyme-peroxidase dimers: Activity and stability.
A facile method for the formation of covalent bonds between protein molecules is zero-length cross-linking. This method enables the formation of cross-links without use of any chemical reagents. Here, the cross-linking is performed for lysozyme, peroxidase (a glycoprotein) and between lysozyme-peroxidase by the method of Simons et al. [B.L. Simons, M.C. King, T. Cyr, M.A. Hefford, H. Kaplan, Covalent cross-linking of protein without chemical reagents, Protein Sci. 2002, 11, 1558-1564]. Approximately one-third of the total lysozyme becomes cross-linked and the dimer form was the major product for both enzymes. This modification induced some changes in the kinetic properties of the dimer peroxidase, as evident by two-fold increasing of V(max) compared to the monomer but the enzymatic activity of cross-linked lysozyme dimer was the same as monomer. The activity of lysozyme dimer remained constant up to 10min at 80 degrees C, while peroxidase activity of both monomer and dimer began to decrease after heating. The structural changes of the enzymes were investigated by circular dichroism and intrinsic fluorescence techniques. Near UV result showed lysozyme possess a compact structure in the dimer form but disruption of tertiary structure of peroxidase dimer was observed. Also conformational changes were detected and discussed by intrinsic fluorescence experiments. Effect of several metals in the formation of lysozyme dimer showed that Co(2+) is the most effective one but its effect was marginal. At the end formation of heterogeneous dimer, peroxidase-lysozyme, was achieved using this method.
Yaghoubi
Hashem
H
Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran.
Khajeh
Khosro
K
Hosseinkhani
Saman
S
Ranjbar
Bijan
B
Naderi-Manesh
Hossein
H
D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D019281:Dimerization; D004531:Eggs; D004795:Enzyme Stability; D005260:Female; D005612:Freeze Drying; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D003432:Cross-Linking Reagents; D006735:Horseradish Peroxidase; D009113:Muramidase