Immunoaffinity purification and fluorescence studies of human adenosine deaminase.
Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.
D000243:Adenosine Deaminase / Q000302:isolation & purification* / Q000378:metabolism; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009700:Nucleoside Deaminases / Q000302:isolation & purification*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013950:Thymus Gland / Q000201:enzymology*; D014364:Tryptophan
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D014364:Tryptophan; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase