Hemoglobin alpha chain isoelectric point shift after treatment with urea, sodium cyanate, succinic anhydride, or diethylene triamine pentaacetic anhydride

A monomeric protein, the hemoglobin alpha chain, was used to compare four p rotocols for conjugation with diethylene triamine pentaacetic (DTPA) anhydr ide. Carbamylation and succinylation were also performed. The isoelectric p oint (pl) was 7.7 for the native protein versus only 5.5 to 7.3 for the fiv e carbamylated derivatives and 4.0 to 7.0 for the six succinylated derivati ves. With carbamylation or succinylation, increasing the molar ratio (agent /protein) was associated with a gradual downward pi shift producing trains of bands. This phenomenon did not occur with DTPA conjugation, whose result s varied with the method used; only one derivate (pI 6.7) was produced by a ll four methods, and multiple fine bands with pH values in the vicinity of 3.6 were seen. For the protein, the pi shift varied with the number of grou ps inserted on the primary amine residues. Also, the shift was larger if th e inserted groups carried electrically-charged moieties.