The denaturation of proteins

THE present paper contains an account of work carried out to determine, with a greater degree of precision than has been hitherto possible, whether or no there is any change in the number of free amino-and carboxyl groups in the protein on denaturation.Let us first consider the work carried out by P. S. Lewis [1927] in this laboratory in which he determined the number of free carboxyl and amino- groups in haemoglobin before and after denaturation.His method was to add a known amount of standard acid or alkali to a definite volume of protein solution of known strength, and then to measure the PH.The PH was then plotted against the amount of acid or alkali added, for denatured and unde- natured protein respectively.He found that the curves were identical, and so concluded that there was no change in the number of free amino-and carboxyl groups as a consequence of denaturation.However, in the light of later work by Cubin [1929], also in this laboratory, on denaturation at low temperatures and low PH values, it appears that the concentration of acid in most of P. S. Lewis's experiments was sufficient to denature the haemoglobin during the time taken in carrying out the titration process itself.Thus from Cubin's results of the rate of denaturation at 250, the temperature at which P. S. Lewis performed his titrations, it is found that denaturation takes place below pH 4-0 almost instantaneously.The titration curve obtained by P. S. Lewis depends on eight points, three of which are above PH 4 and five below.The titration is complete at a PH of about 2-2.Hence we must conclude that in his measurements the amount of acid added for complete titration (obtained from the more acid end of the curve) was the same because he was in each case dealing with denatured protein.This fact has been confirmed by the writer in the following way.A haemoglobin solution of the strength used by P. S. Lewis was kept at 250 in the same quantity of 0096 N acid and for the same length of time as employed by him.The solution was rapidly cooled