Labeling proteins with Tc-99m via hydrazinonicotinamide (HYNIC): Optimization of the conjugation reaction

At present there is considerable interest in labeling peptides with Tc-99m for the development of target specific radiopharmaceuticals for imaging pur poses. In the present study the conjugation of the bifunctional coupling ag ent succinimidyl-hydrazinonicotinamide (S-HYNIC) was studied and optimized in a series of peptides [molecular weight (MW) 6.5-14.3 kDa]. Aprotinin (MW 6.5 kDa), cytochrome C (MW 12,4 kDa), alpha -lactalbumin (MW 14.2 kDa), an d lysozyme (MW 14.3 kDa) were conjugated with S-HYNIC via the epsilon amino groups of their lysine residues. The effects of molar conjugation ratio, r eaction temperature, pH, and protein concentration were studied. Reaction p roducts were analyzed both with respect to the HYNIC-substitution ratio ( s pectrophotometrically) as well as to the labeling efficiency silica gel ins tant thin layer chromatography (SG-ITLC) and molecular size fast performanc e liquid chromatography (FPLC). The effects of conjugation on biological ac tivity were studied in three proteins binding to receptors on leukocytes: i nterleukin-8 (MW 8.5 kDa), interleukin-1 alpha. (MW 17 kDa), and interleuki n-1 receptor antagonist (MW 17 kDa). The labeling efficiency of aprotinin, cytochrome c, alpha -lactalbumin, and lysozyme conjugated under optimal con jugation conditions exceeded 90%, Specific activities obtained were up to 7 .5 MBq/mug. Conjugation was most efficient at 0 degreesC las compared to 20 and 40 degreesC), at pH 8.2 las compared to 6.0, 7.2, and 9.5), and at pro tein concentrations greater than or equal to 2.5 mg/mL. In general, efficie ncy increased with increasing molar conjugation ratio (protein-HYNIC-ratio 1:3 < 1:6 < 1:15 < 1:30). For the receptor binding proteins, biological act ivity was preserved only under the mildest conjugation conditions. For each of these proteins an inverse relation between labeling efficiency and rece ptor binding capacity was found. Labeling proteins with Tc-99m using S-HYNI C is easy, rapid, and efficient, and preparations with high specific activi ty can be obtained. However, biological activity of proteins may be lost at high HYNIC substitution ratios. With the proteins tested here a careful ba lancing of reaction conditions resulted in acceptable, although suboptimal, labeling efficiencies and preservation of biological activity. NUCL MED BI OL 27;6:599-604 2000. (C) 2000 EIsevier Science Inc. All rights reserved.