Interaction of native and apo-carbonic anhydrase with hydrophobic adsorbents: A comparative structure-function study.
Our previous studies indicated that native carbonic anhydrase does not interact with hydrophobic adsorbents and that it acquires this ability upon denaturation. In the present study, an apo form of the enzyme was prepared by removal of zinc and a comparative study was performed on some characteristic features of the apo and native forms by far- and near-UV circular dichroism (CD), intrinsic fluorescent spectroscopy, 1-anilino naphthalene-8-sulfonate (ANS) binding, fluorescence quenching by acrylamide, and Tm measurement. Results indicate that protein flexibility is enhanced and the hydrophobic sites become more exposed upon conversion to the apo form. Accordingly, the apo structure showed a greater affinity for interaction with hydrophobic adsorbents as compared with the native structure. As observed for the native enzyme, heat denaturation of the apo form promoted interaction with alkyl residues present on the adsorbents and, by cooling followed by addition of zinc, catalytically-active immobilized preparations were obtained.
Salemi
Zahra
Z
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Hosseinkhani
Saman
S
Ranjbar
Bijan
B
Nemat-Gorgani
Mohsen
M
D020106:Acrylamide / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry* / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D006361:Heating; D020035:Holoenzymes / Q000737:chemistry* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D011485:Protein Binding; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D001051:Apoenzymes; D020035:Holoenzymes; D020106:Acrylamide; C027132:1-anilino-8-naphthalenesulfonate; D002256:Carbonic Anhydrases; D015032:Zinc