Fluorescein-Isothiocyanated Insulin Interactions with Human Platelets

Abstract

The interactions of fluorescein-isothiocyanated porcine insulin (FITC-insulin) with human platelets were examined by a fluorescence polarization technique. The degree of polarization of the incubation mixture of FITC-insulin and platelets was changed by the ratio of bound to free insulin. This method enabled us to follow the time course of the reaction in the homogeneous assay system without separating the insulin-receptor complex, and it was shown to be useful in the study of insulin binding and dissociation. Insulin binding to trypsinized platelets was considered to be nonspecific, since this fraction remained relatively constant during the incubation period at 25 C. Specific binding was evaluated by subtracting the nonspecific binding from the binding to untreated platelets; specific binding reached a plateau at 60 min. Scatchard analysis was conducted by adding increasing amounts of FITC-insulin to a platelet suspension. Furthermore, some of the prerequisites in analyzing insulin-receptor interaction were examined by this method.

Synthesis and Characterization of Insulin−Fluorescein Derivatives for Bioanalytical Applications

“…Preparation of Insulin − Fluorescein Conjugates. Reaction conditions were a modification of Bromer et al and Shimoyama …”

“…A number of different insulin conjugates have been synthesized and structurally characterized . Specifically, moieties such as fluorophores, − chromophores, biotin, sugars, − saporin, and iodine 27-29 have been chemically conjugated to insulin through the primary amine-, − tyrosine-, − and cysteine-reactive groups …”

“…As mentioned earlier, insulin has been previously labeled with fluorescein isothiocyanate (FITC) and characterized for a number of applications. ,,− The biological activity of the insulin−FITC derivatives has been reported to differ from that of native insulin. , Bromer et al provided the first biological activity information on different insulin−FITC species by performing a mouse convulsion test and immune hemolysis inhibition assay. The activity of a mixture of two monosubstituted insulins was reported to be 40% of native insulin while the di- and trisubstituted species had virtually no biological activity.…”

“…A number of different insulin conjugates have been synthesized and structurally characterized. 8 Specifically, moieties such as fluorophores, [10][11][12][13][14][15][16][17][18][19][20] chromophores, 21 biotin, 22 sugars, [23][24][25] saporin, 26 and iodine [27][28][29] have been chemically conjugated to insulin through † Eli Lilly and Co. ‡ Terrapin Technologies.…”

Platelet sodium-hydrogen antiport in obese and diabetic black women.

“…Insulin receptors are present on platelets and they can be downregulated in diabetes. 36 " 38 However, a controversy exists concerning whether these receptors are coupled to intracellular signals. 39 …”