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A COLORIMETRIC METHOD FOR THE ESTIMATION OF AMINO-ACID a-NITROGEN. II. APPLICATION TO THE HYT)ROLYSIS OF PROTEINS BY PANCREATIC ENZYMES. BY VICTOR JOHN HARDING AND REGINALD M. MACLE AN. (From the Biochem ical Laboratory, McGill University, Montreal.) (Received for pub licatio n, March 2, 1916.) In the first communication on this subject’ the authors showed that the interaction of ami no- acids and triketohydrindene hydr ate (ninhy drin reaction ) in presence of pyridi ne could be used as a mean s of estimati ng amino-acid a-nitrogen calorime trically. The amount of blue coloring matter (ammonium salt of diket ohydr in- dylidene- diketohydrindamine) formed is proportional to the amount of amino-acid a-nitrogen when the l atter is between the limits of 0.05 mg. and 0.005 mg. per cc. of liquid to be estimated. It was claimed that the method could be used as a means of follow- ing protein hydrolysi s and this present paper contains th e e vi- dence in support of such a b elief. We h av e also subjected to criticis m and reexamin ation the mat- ter of our previous paper where it affects the method for our calori metric determination, and a year’s experience with the proc- esshas shown us one o r two points which require amplification. The Standa rd Alani ne Solution.-The standard alanine solution is made as previously directed by dissolving 0.3178 gm. of freshly crystallized alanine in water and making up the volume to 1 liter. Its stability varies a little. Thus one solution was found to be stable for a period of 3 months while a second only ga ve about 95 per cent of the theoretical amount of standar d color at the end of that time. In consequence we have found it advisable to check the stability of our standard alanine b y comparing it from time to time with a fresh ly prepared solution. The Standard Co lor.-We have made several further attempts to prepare a permanent s tandard color, but without succes s. The  b  y  g  u  e  s  t   ,  o  O  c  t   o  b  e r 1  0  , 2  0 1  0 w w w  j   b  c  o r  g D  o w l   o  a  d  e  d f  r  o  

J. Biol. Chem.-1916-Harding-503-17

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