The Labile Amide in Insulin: Preparation of Desalanine-Desamido-Insulin^*

Evidence for a Common Intermediate in Insulin Deamidation and Covalent Dimer Formation: Effects of pH and Aniline Trapping in Dilute Acidic Solutions

“…Therefore, the insulin dimer should be cleaved into five fragments, whereas native insulin should be cleaved into only four fragments. Following incubation with V8, chromatographic separation of the fragments was achieved with the RP300 column, an initial mobile phase of 84% A and 16% B, a gradient 1 rnin after injection (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).4% B at 0.7%/min, 28.4-50% B at 2.2%/ min) with a flow rate of 1.6 mllmin, and UV detection at 214 nm. The fragment that contained the suspected dimer linkage was identified by comparing the digestion patterns of the modified and unmodified insulins.…”

Isolation of a Crystalline A14-(N-Methylpyridinium) Derivative of Bovine Insulin

“…7057). The 2-chloro-jV-methylpyridinium chloride was prepared by the method of Liveris and Miller' 3^ while the 2-iodo derivative was obtained by the procedure of Bradlow and Vanderwerf' 4 l Carrier ampholine (pH [5][6][7][8], used for isoelectric focusing, was obtained from LKB-Produkter, Sweden. All column separations were done at 4 °C.…”

Crystalline [A21-Desamido] Bovine Insulin

“…This buffer was prepared by mixing 12.5 m/ of a 0.2M solution of each constituent and adjusting the pH to 9.4 with l.OM HC1, then diluting to 100 m/ with distilled waterl 15 1 Since the pK of this amine buffer is markedly temperature dependent, the buffer pH was adjusted to the desired value at 30 °d 16 1 Bovine insulin (20 mg) was dissolved in 20 ml of the mixed buffer solution. An aliquot of the enzyme solution containing l/25th of the mass of insulin was added to the insulin solution and hydrolysis allowed to proceed for 8 h at 30 °C.…”