Effect of Calcium Ion on Conformation of Horseradish Peroxidase Isoenzyme C.
Horseradish peroxidase isoenzyme C without ligand [apo-HRP(C)] was studied by fluorescence spectroscopy in this paper. It was observed: 1. there was a distinct Tyr fluorescence in the intrinsic fluorescence of apo-HRP(C), which was seldom observed in other "B" proteins; 2. low concentration denaturants (<2 mol/L urea or 0.2 mol/L GuHCl) could increase the fluorescence intensity of the Trp residue; however they did not change the spectrum characteristics; 3. further increase in the denaturant concentration exposed the Trp residue in HRP(C) to solvent, resulting in a little decrease in fluorescence intensity and a red shift in the spectrum; 4. the similar phenomena were also observed when EDTA, a chelator of calcium, was added to apo-HRP(C). It was also observed that, when the fluorescence of apo-HRP(C) had been affected by either EDTA or low concentration denaturant, it was not more affected by the other one, suggesting that the EDTA and low concentration denaturants might affect Trp fluorescent probe ANS to apo-HRP(C) indicated that the dissociation constants for ANS binding were 15.4 &mgr;mol/L and 7.3 &mgr;mol/L, in the absence and presence of calcium ion, respectively, implying that calcium ion could enhance the binding ability of ANS to the active site of apo-HRP(C), and this suggested that the effect of calcium ion in the enzyme activity might be related to its influence on the conformation of heme binding site in HRP(C).
Wu
Tian-Ping
TP
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. kcruan@sunm.shcnc.ac.cn
Zheng
Le
L
Ruan
Kang-Cheng
KC