The action of nitrous acid on pepsin

IN the search for the source of the specific activity of a protein,such as pepsin one of the few available methods is to act on the protein with reagents which are known to attack cert4in types of group and to see whether the activity changes.Thus Herriott & Northrop [1934] and Herriott have acetylated pepsin with ketene, and Herriott [1937] has iodinated it.The conclusion from both researches was that tyrosine groups played a prominent part in the activity of pepsin.The work here described was started some time ago when the conclusions would have been more novel.Nevertheless it provides further evidence of the participation of tyrosine in the activity of pepsin, and modifies the frequently quoted claim of Hugounenq & Loiseleur [1925] that the activity of pepsin is unaffected by nitrous acid.It also confirms other evidence that only a portion of the tyrosine groups in pepsin can react, at least at rates comparable with that of free tyrosine.Preliminary experiments The earliest experiments were done in collaboration with Miss W. Hessey and Mr B. Mainwaring.(1) Action of NaNO2 and HCI.It was found that 1-7 N NaNO2 plus 1-7 N HC1 added to a solution of crystalline pepsin (about 1 %) at 25° caused rapid formation of a pale yellow precipitate.Activity determinations were uncertain owing to the insolubility of the precipitate, but very little activity could be detected.Hence the statement made by Hugounenq & Loiseleur [1925] that pepsin is not inactivated by nitrous acid "in a medium of hydrochloric acid" does not hold for pure pepsin though it may have held for the crude preparations available at that time.(2) Action of NaNO2 in acetate buffer solutions.It was found that provided that the pH was kept above 3-8 by acetate buffer no precipitate was formed, but the solution slowly became bright yellow and the activity fell off at a measurable rate to about half its original value, with a very much slower subsequent fall to zero.The yellow colour was accompanied by general absorption of light rising continuously from the blue to the short ultra-violet, and quantitative measure- ment showed that the band at 350-60m,u, due to N02-, was not perceptibly diminished in intensity, while that at 275 m,u, due chiefly to tyrosine groups in the pepsin, was diminished, although the total absorptions at both wave- lengths were greatly increased by the yellow substance.This yellow substance is now known to be a diazo compound, since it shows all the colour reactions (coupling in alkaline solution with phenols, production of a red colour on irradiating in alkaline solution, colourless on irradiation in acid solution) characteristic of o-diazo-p-cresol and o-diazodeaminotyrosine, which we have ( 542)