Shashidhara K S, Gaikwad Sushama M
Biochemical Sciences Division, National Chemical Laboratory, Pune, India.
J Fluoresc. 2007 Nov;17(6):599-605. doi: 10.1007/s10895-007-0227-8. Epub 2007 Sep 6.
Apart from the vital role in glycoprotein biosynthesis and degradation, alpha-mannosidase is currently an important therapeutic target for the development of anticancer agents. Fluorescence quenching and time-resolved fluorescence of alpha-mannosidase, a multitryptophan protein from Aspergillus fischeri were carried out to investigate the tryptophan environment. The tryptophans were found to be differentially exposed to the solvent and were not fully accessible to the neutral quencher indicating heterogeneity in the environment. Quenching of the fluorescence by acrylamide was collisional. Surface tryptophans were found to have predominantly positively charged amino acids around them and differentially accessible to the ionic quenchers. Denaturation led to more exposure of tryptophans to the solvent and consequently in the significant increase in quenching with all the quenchers. The native enzyme showed two different lifetimes, tau (1) (1.51 ns) and tau (2) (5.99 ns). The average lifetime of the native protein (tau) (3.187 ns) was not affected much after denaturation (tau) (3.219 ns), while average lifetime of the quenched protein samples was drastically reduced (1.995 ns for acrylamide and 1.537 ns for iodide). This is an attempt towards the conformational studies of alpha-mannosidase.
除了在糖蛋白生物合成和降解中发挥重要作用外，α-甘露糖苷酶目前是抗癌药物开发的重要治疗靶点。对来自费氏曲霉的一种多色氨酸蛋白α-甘露糖苷酶进行了荧光猝灭和时间分辨荧光研究，以探究色氨酸环境。发现色氨酸对溶剂的暴露程度不同，中性猝灭剂无法完全接近它们，这表明环境存在异质性。丙烯酰胺对荧光的猝灭是碰撞性的。发现表面色氨酸周围主要是带正电荷的氨基酸，且对离子猝灭剂的可及性不同。变性导致色氨酸更多地暴露于溶剂中，因此所有猝灭剂的猝灭作用都显著增加。天然酶表现出两种不同的寿命，τ(1)（1.51纳秒）和τ(2)（5.99纳秒）。变性后天然蛋白的平均寿命（τ）（3.187纳秒）变化不大（τ）（3.219纳秒），而猝灭蛋白样品的平均寿命则大幅缩短（丙烯酰胺为1.995纳秒，碘化物为1.537纳秒）。这是对α-甘露糖苷酶构象研究的一次尝试。
