Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium-HCl, urea, and pH.


Abstract

The stability of bovine lactoperoxidase to denaturation by guanidinium-HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near Tm is controlled by kinetics. The values for the thermal transition, Tm, at slow heating scan rate were 66.8, 61.1, and 47.2 degrees C in the presence of 0.5, 1, and 2 M guanidinium-HCl, respectively. The extrapolated value for Tm in the absence of guanidinium-HCl is 73.7 degrees C, compared with 70.2 degrees C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, Cp, at Tm and Ea for the thermal transition decrease under conditions where Tm is lowered. At a given concentration, urea is less effective than guanidinium-HCl in reducing Tm, but urea reduces Cp relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 degrees C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164-1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change. Study holds ProTherm entries: 25720, 25721, 25722, 25723, 25724, 25725, 25726, 25727, 25728, 25729, 25730, 25731, 25732, 25733, 25734, 25735, 25736, 25737, 25738, 25739, 25740, 25741, 25742 Extra Details: Scan rate: 0.25 degrees/min Protein stability; Denaturation

Submission Details

ID: wCVpmdKo

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:56 p.m.

Version: 1

Publication Details
Zelent B;Sharp KA;Vanderkooi JM,Biochim. Biophys. Acta (2010) Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium-HCl, urea, and pH. PMID:20298816
Additional Information

Number of data points 43
Proteins Lactoperoxidase ; Lactoperoxidase
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dCp pH:8.9, details:Additives 1M GdnHCl ; Experimental Assay: Tm pH:8.9, details:Additives 1M GdnHCl ; Experimental Assay: dCp details:Additives 1M GdnHCl, pH:8.0 ; Experimental Assay: Tm details:Additives 1M GdnHCl, pH:8.0 ; Experimental Assay: dCp details:Additives 1M GdnHCl, pH:7.0 ; Experimental Assay: Tm details:Additives 1M GdnHCl, pH:7.0 ; Experimental Assay: dCp details:Additives 1M GdnHCl, pH:5.1 ; Experimental Assay: Tm details:Additives 1M GdnHCl, pH:5.1 ; Experimental Assay: dCp details:Additives 1M GdnHCl, pH:4.1 ; Experimental Assay: Tm details:Additives 1M GdnHCl, pH:4.1 ; Experimental Assay: dCp pH:6.0, details:Additives 2M urea ; Experimental Assay: Tm pH:6.0, details:Additives 2M urea ; Experimental Assay: dCp pH:6.0, details:Additives 1M urea ; Experimental Assay: Tm pH:6.0, details:Additives 1M urea ; Experimental Assay: dCp pH:6.0, details:Additives 2M GdnHCl ; Experimental Assay: Tm pH:6.0, details:Additives 2M GdnHCl ; Experimental Assay: dCp pH:6.0, details:Additives 1M GdnHCl ; Experimental Assay: Tm pH:6.0, details:Additives 1M GdnHCl ; Experimental Assay: dCp pH:6.0, details:Additives 0.5M GdnHCl ; Experimental Assay: Tm pH:6.0, details:Additives 0.5M GdnHCl ; Experimental Assay: dCp pH:6.0, details:Additives ; Experimental Assay: Tm pH:6.0, details:Additives
Libraries Mutations for sequence VKCDENSPYRTITGDCNNRRSPALGAANRALARWLPAEYEDGLALPFGWTQRKTRNGFRVPLAREVSNKIVGYLDEEGVLDQNRSLLFMQWGQIVDHDLDFAPETELGSNEHSKTQCEEYCIQGDNCFPIMFPKNDPKLKTQGKCMPFFRAGFVCPTPPYQSLAREQINAVTSFLDASLVYGSEPSLASRLRNLSSPLGLMAVNQEAWDHGLAYLPFNNKKPSPCEFINTTARVPCFLAGDFRASEQILLATAHTLLLREHNRLARELKKLNPHWNGEKLYQEARKILGAFIQIITFRDYLPIVLGSEMQKWIPPYQGYNNSVDPRISNVFTFAFRFGHMEVPSTVSRLDENYQPWGPEAELPLHTLFFNTWRIIKDGGIDPLVRGLLAKKSKLMNQDKMVTSELRNKLFQPTHKIHGFDLAAINLQRCRDHGMPGYNSWRGFCGLSQPKTLKGLQTVLKNKILAKKLMDLYKTPDNIDIWIGGNAEPMVERGRVGPLLACLLGRQFQQIRDGDRFWWENPGVFTEKQRDSLQKVSFSRLICDNTHITKVPLHAFQANNYPHDFVDCSTVDKLDLSPWASREN
Sequence Assay Result Units