Lee K C, Kang T S, Woo B H, Lee J T, Lee H S, DeLuca P P
College of Pharmacy, SungKyunKwan University, Jangan-ku, Suwon City, South Korea.
J Chromatogr B Biomed Sci Appl. 1997 Jun 20;694(1):31-7. doi: 10.1016/s0378-4347(97)00154-0.
Reversed-phase HPLC conditions for simultaneous separation of salmon calcitonin, mono- and di-radioiodinated salmon calcitonins and their tryptic digested fragments have been developed. Salmon calcitonin was radioiodinated with Na125I by the iodo-beads method. After solid-phase extraction from the reaction mixtures using C18 Bond Elut cartridges, mono- and di-radioiodinated salmon calcitonins were separated from each other, as well as from unlabeled salmon calcitonin, on a Bondclone 10 C18 column (300x7.8 mm I.D.) by isocratic elution with 0.1% trifluoroacetic acid in 34% aqueous acetonitrile. The characteristics of either iodinated peptides or unlabeled salmon calcitonin were evaluated on the basis of UV absorbance (215 and 280 nm), fluorescence (lambda(ex)=282 nm, lambda(em)=310 nm) and measurement of specific radioactivity by means of a flow-through radio-isotope detector. HPLC separation of a tryptic digest of iodinated salmon calcitonin fraction on a W-porex 5 C18 300 A column (250x4.6 mm I.D.) and subsequent amino acid analysis, led to the conclusion that radioiodination took place at the Tyr residue and not at the His moiety.
已开发出反相高效液相色谱条件，用于同时分离鲑鱼降钙素、单放射性碘标记和双放射性碘标记的鲑鱼降钙素及其胰蛋白酶消化片段。采用碘珠法用Na125I对鲑鱼降钙素进行放射性碘标记。使用C18 Bond Elut柱从反应混合物中进行固相萃取后，在Bondclone 10 C18柱（内径300x7.8 mm）上，通过在34%乙腈水溶液中用0.1%三氟乙酸等度洗脱，将单放射性碘标记和双放射性碘标记的鲑鱼降钙素彼此分离，同时与未标记的鲑鱼降钙素分离。基于紫外吸光度（215和280 nm）、荧光（激发波长λ(ex)=282 nm，发射波长λ(em)=310 nm）以及通过流通式放射性同位素检测器测量比放射性，对碘化肽或未标记的鲑鱼降钙素的特性进行了评估。在W-porex 5 C18 300 A柱（内径250x4.6 mm）上对碘化鲑鱼降钙素馏分的胰蛋白酶消化产物进行高效液相色谱分离，随后进行氨基酸分析，得出放射性碘标记发生在Tyr残基而非His部分的结论。
