Molten globule-like state of bovine carbonic anhydrase in the presence of acetonitrile.
We have evaluated the effects of acetonitrile on the structure and function of bovine carbonic anhydrase II. The potential structural and functional changes in carbonic anhydrase in the presence of different acetonitrile/buffer ratios (0%, 17.5% and 47.5% v/v) were determined using a variety of methods. These included simple spectrophotometric methods to record enzyme velocity, fluorescence measurements and calculation of accessible surface area (ASA) to identify possible alterations in tertiary structure of the protein, CD measurements to search for secondary structure conversions, and thermal scanning to determine structural stability of the protein in different media. The Far-UV CD studies indicated that carbonic anhydrase, for the most part, retains its secondary structure in the presence of acetonitrile. Fluorescence measurements using iodide ion and ANS along with ASA calculations revealed that in the presence of acetonitrile some degree of conformational change occurs in the carbonic anhydrase structure. In addition to the hydrophobic pockets, two additional tryptophanyl residues become exposed to the solvent, thereby increasing the surface hydrophobicity of the protein. These alterations dramatically reduce the catalytic activity, thermal stability, and aggregation velocity of the enzyme. Thus, our results support a molten globule-like structure of carbonic anhydrase in the presence of acetonitrile.
Safarian
Shahrokh
S
School of Biology, University College of Science, University of Tehran, Iran. safarian@ibb.ut.ac.ir
Saffarzadeh
Mona
M
Zargar
Sayyed Jalal
SJ
Moosavi-Movahedi
Ali Akbar
AA
D000097:Acetonitriles / Q000494:pharmacology*; D000818:Animals; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D002417:Cattle; D002942:Circular Dichroism / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability / Q000187:drug effects; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D000097:Acetonitriles; D014364:Tryptophan; D024402:Carbonic Anhydrase II; D002256:Carbonic Anhydrases; C032159:acetonitrile