Puri R N, Roskoski R
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans 70119.
Anal Biochem. 1988 Aug 15;173(1):26-32. doi: 10.1016/0003-2697(88)90153-4.
o-Phthalaldehyde has been recently shown to be a useful reagent for chemical modification of cyclic nucleotide dependent protein kinases, hexokinase, and fructose-1,6-bisphosphatase. It reacts covalently with closely spaced (approximately 3 A) sulfhydryl and epsilon-amino functions of cysteine and lysine residues, respectively, of these enzymes to yield fluorescent isoindole derivatives. We have found the reagent to be equally useful to investigate the degree of reactivity of sulfhydryl and amino functions in substances that do not possess enzymatic activity, e.g., glutathione, homocysteine, and cysteine. The kinetics of the reaction of nonenzymatic aminothiols with o-phthalaldehyde can be followed rapidly and conveniently by continuously monitoring the increase in relative fluorescence of the isoindole derivatives. The fluorescence emission maxima of the o-phthalaldehyde adducts can be used to compute molar transition energies that provide qualitative but useful information concerning the degree of polarity of microenvironment of the sulfhydryl and amino functions participating in isoindole formation. The kinetic and spectral data obtained from the reaction between o-phthalaldehyde and nonenzymatic low molecular weight aminothiols may be helpful in comparing the reactivities of the sulfhydryl and amino functions in enzymes.
邻苯二甲醛最近被证明是一种用于化学修饰环核苷酸依赖性蛋白激酶、己糖激酶和果糖-1,6-二磷酸酶的有用试剂。它分别与这些酶中半胱氨酸和赖氨酸残基间距相近（约3埃）的巯基和ε-氨基功能基团发生共价反应，生成荧光异吲哚衍生物。我们发现该试剂对于研究不具有酶活性的物质（如谷胱甘肽、同型半胱氨酸和半胱氨酸）中巯基和氨基功能基团的反应程度同样有用。通过连续监测异吲哚衍生物相对荧光的增加，可以快速方便地跟踪非酶促氨基硫醇与邻苯二甲醛的反应动力学。邻苯二甲醛加合物的荧光发射最大值可用于计算摩尔跃迁能，这些能量提供了有关参与异吲哚形成的巯基和氨基功能基团微环境极性程度的定性但有用的信息。从邻苯二甲醛与非酶促低分子量氨基硫醇反应中获得的动力学和光谱数据可能有助于比较酶中巯基和氨基功能基团的反应活性。
