Studies on the polarography of proteins. 2. The influence of pH and concentrations of ammonia and ammonium ion upon the polarographic waves

Abstract

An investigation was made of (a) the effect of electrolyte pH upon the 1st 2 polarographic protein waves of 5 crystalline proteins when the total molarities of the ammonia buffer and Co were maintained constant, and of (b) the effects upon the 3 polarographic protein waves of crystalline bovine plasma albumin of varying the concns. of NH3 and of NH4+ at nearly constant electrolyte pH. Under the conditions of (a) the wave heights rose when the electrolyte pH was raised from about pH 3.5 to 8.5 if Co++ was used. When Co+++ was used, the wave heights in neutral and acid media (ia) were quite constant. It is concluded that the enhancement in Co++ soln. is due to the concomitant increase in concn. of cobaltous hexammine ion. There is a strong correlation between ia and the total potential sulfydryl content of these proteins. This is the 1st time that a quantitative relationship has been shown between protein waves I and II and the amino acid composition of a protein. A satisfactory explanation of this relationship cannot be offered, but it implies that all the potential sulfydryl groups of the protein may be reactive in acid media. In media poised at neutral or acid pH, the pH of the soln. layer surrounding the Hg drop ( the reaction layer) is probably more alkaline than the bulk of the electrolyte. In a Co+++ soln. the pH of the reaction layer is indicated by the pH wave height curve of a particular protein. The protein waves are enhanced when the pH of the electrolyte is in the neighborhood of the pK of the buffer system and they attain a max. value on the alkaline side of the buffer pK. At high pH the waves are depressed. The pH at which the max. wave height occurs seems to depend upon the tyrosine content and the arginine plus lysine content of the protein. The magnitude of the max. wave height is related to the total potential sulfydryl content of the protein, but the relationship is not linear. Certain evidence is tentatively interpreted to mean that at high electrolyte pH the wave height is limited by the dissociation of sulfydryl groups. Under the conditions of (b) waves I and II are enhanced when the total molarity of the buffer is increased or when the concn. of one of the buffer components is maintained constant and the other is increased. Under these conditions the wave heights are independent of pH in the range 8.7-9.5. Brdicka''s observations on the effect of pH on wave III, the "prenatrium" wave, were confirmed for conditions of low buffer molarity. The effect seems to be more complex when the buffer concn. is high. The reactions and equilibria responsible for the effects are discussed, emphasis being laid on the importance of cobaltous hexammine ion in the catalytic reactions.