Reversed-phase high-performance liquid chromatography of radioiodinated salmon calcitonins.
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.
Lee
K C
KC
College of Pharmacy, SungKyunKwan University, Jangan-ku, Suwon City, South Korea.
Kang
T S
TS
Woo
B H
BH
Lee
J T
JT
Lee
H S
HS
DeLuca
P P
PP
D000595:Amino Acid Sequence; D002116:Calcitonin / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry
D007457:Iodine Radioisotopes; C028815:salmon calcitonin; D002116:Calcitonin; D014357:Trypsin