Stoyanov Alexander V, Ahmadzadeh Hossein, Krylov Sergey N
Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3.
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Nov 25;780(2):283-7. doi: 10.1016/s1570-0232(02)00535-4.
Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid-base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern--the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.
当需要高灵敏度荧光检测时，荧光试剂用于蛋白质标记。与使用活性荧光染料（如异硫氰酸荧光素）的传统标记类似，荧光反应预计会改变蛋白质的物理化学性质。了解这些变化对于有效分离和鉴定标记蛋白质可能至关重要。在这里，我们研究了用荧光试剂标记肌红蛋白对蛋白质酸碱性质的影响。所用的荧光试剂是3-(2-呋喃甲酰基)喹啉-2-甲醛（FQ）。在平板凝胶等电聚焦中，我们发现标记反应产生了至少六种等电点值低于未标记肌红蛋白的物种。这些物种可被鉴定为肌红蛋白与一至六个FQ分子逐步标记的产物。当通过毛细管区带电泳分析反应产物时，观察到了相同系列的FQ标记物种。通过比较实验和理论等电点值，我们能够阐明标记模式——通过等电聚焦检测到的每种标记产物对应的FQ分子数。
