PROTEINS 91 



calculation of the weight of nitrogen gas corresponding to the volume obtained is 

 most readily made with the aid of the tables (see page 90) devised for this purpose. 1 



The Van Slyke Micro-apparatus. 2 In later work Van Slyke has used to a large 

 extent an apparatus which differs from the one described above only in being con- 

 siderably smaller. More accurate measurements can be made with this and 

 smaller amounts of ammo nitrogen determined. In using this only 10 c.c. of 

 nitrite solution and 2.5 c.c. of acetic acid are required for an analysis. One-fifth 

 the amount of substance may be analyzed with the same degree of accuracy as 

 with the larger apparatus. Practically the only alteration from the mode of opera- 

 tion already detailed above, is in the speeds at which the deaminizing bulb and the 

 Hempel pipette are shaken. During the first stage of the analysis the deaminizing 

 bulb should be shaken by the motor at a very high rate of speed, about as fast as 

 the eye can follow or an unnecessary amount of time is lost in freeing the apparatus 

 from air. This stage is also much accelerated by warming the nitrite solution to 

 30 before it is used, in case a low room temperature has reduced the temperature 

 of the solutions below 20. In the third stage when the nitric oxide is being ab- 

 sorbed by the permanganate, the Hempel pipette should be shaken not faster than 

 twice per second. This is to prevent the breaking off of small gas bubbles. 



It is especially necessary that in the first stage the removal of air be complete. 

 This is assured by shaking the solution in the deaminizing bulb back each time, 

 in this stage, until the bulb is two-thirds filled with nitric oxide. 



For the determination of total and free amino acid nitrogen in the urine by 

 this method see chapter on Quantitative Analysis of Urine. 



ESTIMATION OF AMINO-ACID -NITROGEN 



Method of Harding and MacLean. 3 Principle. Amino-acid mixtures when 

 treated with triketohydrindene hydrate give a colored solution which may be com- 

 pared colorimetrically with a standard. 



Procedure. One c.c. of the solution to be estimated (containing not more than 

 0.05 mg. of ammo-acid a-nitrogen and neutral to phenolphthalein, is mixed with 

 i c.c. of a 10 per cent aqueous solution of pure pyridine and i c.c. of a freshly pre- 

 pared 2 per cent solution of triketohydrindene hydrate and heated in a rapidly 

 boiling constant-level water-bath for 20 minutes. At the end of that time the test 

 tube is removed, cooled and diluted to a suitable volume, usually 100 c.c., but if the 

 amino-acid a-nitrogen is very small in amount a correspondingly smaller dilution 

 can be used. The solution is compared with a standard in a Duboscq colorimeter. 

 The standard solution is prepared by dissolving 0.3178 gm. of pure, freshly crystal- 

 lized alanine in a liter of distilled water. The solution contains 0.05 mg. of N per 

 c.c. Treat i c.c. of this standard just as above, except that only i c.c. of trike- 

 tohydrindene is required. The standard solution is stable for three months. 

 Amounts of amino nitrogen from 0.005 to 0.05 mg. may be determined. The 

 method is inaccurate for cystine and has not yet been adapted for use with biolog- 

 ical fluids other than solutions of protein hydrolysis products. 



1 See Van Slyke: Jour. Biol. Chem., 12, 275, 1912 or Gattermann: Praxis des organis- 

 chen Chemikers, ninth edition. In using the tables in the latter work or similar tables it 

 should be borne in mind that the volume of nitrogen gas must be divided by two, inasmuch 

 as only one-half of the nitrogen collected comes from the amino groups. 



2 Either of these apparatus may be obtained from Emil Greiner, 45 Cliff Street, New 

 York, or from Robert Goetze, Leipzig. Van Slyke has recently described a third form of 

 his apparatus about half of the size of the earlier micro-apparatus. This has a more 

 accurate burette so that the gas volumes can be read to o.ooi c.c. (Van Slyke: Jour. Biol. 

 Chem., 23, 407, 1915.) 



