Analytical Sciences

Heme-Undecapeptide Labeling on Insulin for the Immunoassay of Insulin with Chemiluminescence Detection

Kiyoshi ZAITSU * , Yoshitada KIMURA, Yoshihito OHBA, Kenji HAMASE, Yuji MOTOMURA, Minoru ITOSE and Masato ISHIYAMA

Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi, Fukuoka 812-8582, Japan

Heme-undecapeptide (HUP, microperoxidase-11) labeled insulins, in which the Lys(3) amino group of HUP was crosslinked to one amino group of insulin (Gly(A1), Phe(B1) or Lys(B29)) via a disulfide linkage, were prepared. Lys(3)-[3-(2-pyridyldithio) propionoyl]-HUP (Lys(3)-PDP-HUP) was synthesized by the reaction of HUP with N-succinimidyl 3-(2-pyridyldithio)propionate and then conjugated with Gly(A1)-thioglycoloyl- (Gly(A1)-TG-), Phe(B1)-TG- or Lys(B29)-TG-insulin. Each TG-insulin was generated by deacetylation of Gly(A1)-acetylthioglycoloyl-(Gly(A1)-ATG-), Phe(B1)-ATG- or Lys(B29)-ATG-insulin, that was prepared by reacting native insulin, Phe(B1)-3,4,5,6-tetrahydrophthalyl-insulin (Phe(B1)-THP-insulin) or Gly(A1),Phe(B1)-(THP)2-insulin with N-succinimidyl S-acetylthioacetate (SATA), respectively, followed by deprotection of THP, a reversible amino-protecting group. Both preparative reversed-phase and anion-exchange HPLC were used in the syntheses of PDP-HUP and the insulin derivatives (THP-, ATG- and HUP-insulin), respectively. The optimal reaction conditions for these syntheses were extensively studied using HPLC separation. In addition, immunoreactivity of Phe(B1)-HUP-insulin was manifested by using a solid-phase antibody method with chemi-luminescence detection. (Keywords: Heme-undecapeptide labeling, insulin, chemiluminescence, immunoassay)

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