title: Laser photobleaching leads to a fluorescence grade adenosine deaminase.
abstract: The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.
authors.affiliation: Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva, Israel.
authors.forename: A H
authors.initials: AH
authors.lastname: Parola
authors.forename: V R
authors.initials: VR
authors.lastname: Caiolfa
authors.forename: I
authors.initials: I
authors.lastname: Bar
authors.forename: S
authors.initials: S
authors.lastname: Rosenwaks
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives; D000243:Adenosine Deaminase / Q000302:isolation & purification* / Q000528:radiation effects; D058892:Adenosine Deaminase Inhibitors; D007700:Kinetics; D007834:Lasers; D009700:Nucleoside Deaminases / Q000302:isolation & purification*; D010777:Photochemistry; D011048:Polarography; D013050:Spectrometry, Fluorescence
chemical_list: D058892:Adenosine Deaminase Inhibitors; C010192:9-(2-hydroxy-3-nonyl)adenine; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase; D000225:Adenine