Interactions between carbonic anhydrase and its inhibitors revealed by gel electrophoresis and circular dichroism

Gianazza, Elisabetta; Sirtori, Cesare R.; Castiglioni, Silvia; Eberini, Ivano; Chrambach, Andreas; Rondanini, Andrea; Vecchio, Giuseppe

doi:10.1002/(sici)1522-2683(20000501)21:8<1435::aid-elps1435>3.0.co;2-e

Cited by 4 publications

(6 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Binding with FA stabilizes BLGB against urea and SDS denaturations. These stabilizing effects are much less effective than observed for enzyme-inhibitor complexes (44) and confirm that hydrophobic interactions between FA and BLG are not very strong.…”

Section: Discussionsupporting

confidence: 58%

pH and Ionic Strength Dependence of Protein (Un)Folding and Ligand Binding to Bovine β-Lactoglobulins A and B

et al. 2002

Self Cite

View full text Add to dashboard Cite

Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).

show abstract

Section: Discussionsupporting

confidence: 58%

pH and Ionic Strength Dependence of Protein (Un)Folding and Ligand Binding to Bovine β-Lactoglobulins A and B

et al. 2002

Self Cite

View full text Add to dashboard Cite

show abstract

“…427 Although the molar ellipticity of CA in the far-UV region is significantly higher than in the 300 nm region, the binding of ligands to CA leads to minor changes in the spectra in the 200-230 nm region; under the same conditions, the CD bands 280-300 nm change not only shape but also sign. 427 One must, however, approach the interpretation of such results with caution. Most arylsulfonamide ligands exhibit moderate or strong absorption in the near-UV region, and hence, the observed spectral changes may result from changes in either the environment of the aromatic residues or from the induced CD (ICD) signal of the ligand.…”

Section: Magnetic Resonance Spectroscopymentioning

confidence: 99%

“…The rotatory strength of proteins is extremely sensitive to alterations in their secondary structure. Circular dichroism (CD) is, therefore, a useful tool for assaying binding. − The optical activity of CA in the far-UV region (λ < ∼230 nm) results from electronic transitions in the amide bonds, while absorption of the aromatic residues is responsible for the ellipticity observed in the near- and middle-UV (about 320 and 240 nm). Gianazza et al reported an extensive study on binding of acetazolamide ( 137 ), dorzolamide ( 156 ), and methazolamide ( 138 ) to BCA II in the presence and absence of sodium dodecyl sulfate (SDS) .…”

Section: 36 Circular Dichroism Spectroscopymentioning

confidence: 99%

“…Circular dichroism (CD) is, therefore, a useful tool for assaying binding. − The optical activity of CA in the far-UV region (λ < ∼230 nm) results from electronic transitions in the amide bonds, while absorption of the aromatic residues is responsible for the ellipticity observed in the near- and middle-UV (about 320 and 240 nm). Gianazza et al reported an extensive study on binding of acetazolamide ( 137 ), dorzolamide ( 156 ), and methazolamide ( 138 ) to BCA II in the presence and absence of sodium dodecyl sulfate (SDS) . Although the molar ellipticity of CA in the far-UV region is significantly higher than in the 300 nm region, the binding of ligands to CA leads to minor changes in the spectra in the 200−230 nm region; under the same conditions, the CD bands ∼280−300 nm change not only shape but also sign .…”

Section: 36 Circular Dichroism Spectroscopymentioning

confidence: 99%

See 1 more Smart Citation

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

et al. 2008

View full text Add to dashboard Cite

I. Introduction to Carbonic Anhydrase (CA) and to the Review 948 1. Introduction: Overview of CA as a Model 948 1.1. Value of Models 950 1.2. Objectives and Scope of the Review 950 2. Overview of Enzymatic Activity 950 3. Medical Relevance 951 II. Structure and Structure−Function Relationships of CA 953 4. Global and Active-Site Structure 953 4.1. Structure of Isoforms 953 4.2. Isolation and Purification 954 4.3. Crystallization 954 4.4. Structures Determined by X-ray Crystallography and NMR 955 4.4.1. Structures Determined by X-ray Crystallography 955 4.4.2. Structure Determined by NMR 955 4.5. Global Structural Features 963 4.6. Structure of the Binding Cavity 964 4.7. Zn II -Bound Water 965 5. Metalloenzyme Variants 966 6. Structure−Function Relationships in the Catalytic Active Site of CA 968 6.1. Effects of Ligands Directly Bound to Zn II 968 6.2. Effects of Indirect Ligands 969 7. Physical-Organic Models of the Active Site of CA 969 III. Using CA as a Model to Study Protein−Ligand Binding 970 8. Assays for Measuring Thermodynamic and Kinetic Parameters for Binding of Substrates and Inhibitors 970 8.1. Overview 970 8.

show abstract

“…The equilibria A n nA or A n B m nAjmB for dissociation, and A folded A unfolded for denaturation, are driven to the left mostly by enthalpic factors and to the right by entropic factors. Accordingly, holoproteins are usually more stable against denaturation than their apo counterparts (as with transferrin [15], ovotransferrin [16], α-lactoglobulin [17] and carbonic anhydrase [18]).…”

Section: Introductionmentioning

confidence: 99%

Size is a major determinant of dissociation and denaturation behaviour of reconstituted high-density lipoproteins

Gianazza

Eberini

Sirtori

et al. 2002

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

Lipid-free apolipoprotein A-I (apoA-I) and A-I(Milano) (A-I(M)) were compared for their denaturation behaviour by running across transverse gradients of a chaotrope, urea, and of a ionic detergent, SDS. For both apo A-I and monomeric apoA-I(M) in the presence of increasing concentrations of urea the transition from high to low mobility had a sigmoidal course, whereas for dimeric A-I(M)/A-I(M) a non-sigmoidal shape was observed. The co-operativity of the unfolding process was lower for dimeric A-I(M)/A-I(M) than for apoA-I or for monomeric apoA-I(M). A slightly higher susceptibility to denaturation was observed for dimeric A-I(M)/A-I(M) than for monomeric apoA-I(M). A similar behaviour of A-I(M)/A-IM versus apoA-I(M) was observed in CD experiments. Large- (12.7/12.5 nm) and small- (7.8 nm) sized reconstituted high-density lipoproteins (rHDL) containing either apoA-I or A-I(M)/A-I(M) were compared with respect to their protein-lipid dissociation behaviour by subjecting them to electrophoresis in the presence of urea, of SDS and of a non-ionic detergent, Nonidet P40. A higher susceptibility to dissociation of small-sized versus large-sized rHDL, regardless of the apolipoprotein component, was observed in all three instances. Our data demonstrate that the differential plasticity of the various classes of rHDL is a function of their size; the higher stability of 12.5/12.7 nm rHDL is likely connected to the higher number of protein-lipid and lipid-lipid interactions in larger as compared with smaller rHDL.

show abstract

Interactions between carbonic anhydrase and its inhibitors revealed by gel electrophoresis and circular dichroism

Cited by 4 publications

References 37 publications

pH and Ionic Strength Dependence of Protein (Un)Folding and Ligand Binding to Bovine β-Lactoglobulins A and B

pH and Ionic Strength Dependence of Protein (Un)Folding and Ligand Binding to Bovine β-Lactoglobulins A and B

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

Size is a major determinant of dissociation and denaturation behaviour of reconstituted high-density lipoproteins

Contact Info

Product

Resources

About