Parola A H, Caiolfa V R, Bar I, Rosenwaks S
Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva, Israel.
Anal Biochem. 1989 Sep;181(2):383-8. doi: 10.1016/0003-2697(89)90259-5.
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.
来自小牛肠黏膜的腺苷脱氨酶（腺苷氨基水解酶，EC 3.5.4.4）可通过商业途径获得高纯度产品。然而，在可进行荧光研究的灵敏度下，在激发波长最大值（λexmax）为350 nm、发射波长最大值（λemmax）为420 nm时，可检测到一种非肽类荧光。用Nd:YAG激光的三次谐波（355 nm）以5 mJ/脉冲、脉冲宽度为6 ns、重复频率为10 Hz照射16分钟后，这种非肽类荧光降低了七倍。荧光的下降伴随着可忽略不计的酶活性损失。此外，通过以下方式确定了蛋白质的完整性：（i）其荧光（λexmax = 305 nm，λemmax = 335 nm）和寿命分布，以及（ii）其在底物腺苷和两种抑制剂存在下的动力学，所有这些基本上都保持不变。激光光漂白是获得荧光级腺苷脱氨酶的一种简单方法。
