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A modified type of isolectric focusing has been applied successfully to the separation of hemoglobin A1C (HbA1C) from HbA in normal and diabetic cell lysates. It consists of transforming a linear pH gradient into a nonlinear one, by the addition of an amphoteric substance ("separator" or "pH gradient modifier") with an isoelectric point (pI) close to the pI's of the two hemoglobins. Among the "modifiers" tested, histidine, proline, threonine, beta-alanine, 6-amino caproic acid, and 5-amino valeric acid are not useful in the hemoglobin pI range (pH 6.9--7.0). The dipeptide histidyl-glycine (pI = 6.8; pI-pK1 = 1) is very efficient in flattening the pH gradient, in the hemoglobin region. even when added in low concentrations (10--100 mM), thus affording full resolution of the two hemoglobin species.
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