The determination of the isoelectric and isoionic points of haemoglobin from measurements of membrane potentials

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Abstract
The possibility of determining the isoelectric point from measurements of membrane potentials was studied; the most accurate results were obtained when conc. solutions of proteins were equilibrated with dilute buffers at 0[degree]. To facilitate comparisons of isoelectric and isoionic point at which the protein neither takes up nor gives off H ions, methods were developed for the estimation of the isoionic point from the distribution of salts across membranes. At 0[degree], the isoionic point of crystallized Hb (sheep) was at pH 7.6; un-crystallized protein gave pH 7.45 [plus or minus]0.1. In ammonium phosphate buffers of ionic strengths 0.02, 0.04 and 0.07, the isoelectric points of crystallized protein were 7.16, 6.91 and 6.70 respectively. Uncrystallized Hb (7 preparations) , with buffers of ionic strengths 0.02, gave an isoelectric point 7.06 [plus or minus] 0.01. The isoelectric points became more acid as the salt conc. increased; the effects of increasing conc. of chlorides were less than those of phosphates. The min. of osmotic pressure appeared to be at pH values between the isoionic and isoelectric points. The distribution of ammonia and of phosphoric acid between protein crystals, protein solution and dialysate was investigated. It is suggested that the effects of salts on the isoelectric point, and the effects of proteins on the activities of diffusible ions, may be due to a reaction of the type +Pr + Na++ Cl- = [ClPr]- + Na+, where +Pr- represents a zwitterion.