ARGINASE 



20 



ARGININE REACTION 



5% aq. sodium hyposulphite. Counter- 

 stain with alum carmine, mount in 

 usual way. To make Fontana's fluid 

 add ammonia drop by drop to 5% aq. 

 silver nitrate until ppt. formed is ex- 

 actly redissolved; then carefully drop 

 by drop 5% aq. silver nitrate until 

 appearance of persistent cloudiness and 

 the liquid does not smell of ammonia. 

 Decant before employing. See also 

 Clara, M., and Canal, F., Zeit. f. Zellf. 

 u. Mikr. Anat., 1932, 15, 801-808; Clara, 

 M., Ergeb. d. Anat. u. Entw., 1933, 30, 

 240-340. 

 Arginase. It is possible to localize arginase 

 in the cytoplasm and nuclei of liver cells 

 by Behren's technique (Zeit. Physiol. 

 Chem., 1939, 258, 27-32). Finely ground 

 tissue is dried to powder in frozen condi- 

 tion. It is then suspended and cen- 

 trifuged in different mixtures of benzene 

 and carbon tetrachloride. The nuclei 

 only are found in the lowest layer, next 

 comes nuclear debris and above this 

 cytoplasmic debris. His analysis 

 showed arginase present in the same con- 

 centration in the nuclei as in the cyto- 

 plasm. Blaschko and Jacobson (Bourne, 

 p. 217) remark that this is the first in- 

 stance of the demonstration of an enzyme 

 in the cell nucleus. 

 Arginine Reaction. The method of Serra, 

 J. A., Stain Techn., 1946, 21, 5-18 is 

 detailed by him as follows: Prepare 

 tissue as described under Ninhydrin 

 Reaction. 



"1. Before the reaction the pieces or 

 sections are hardened with 10% for- 

 maldehyde during 12-24 hours, the 

 formalin being afterwards well washed 

 out. (If the fixative contains formalin 

 this step can be omitted.) 



"2. Immerse the pieces for 15 minutes 

 in a mixture consisting of 0.5 ml. of 

 diluted a-naphthol ; 0.5 ml. of N NaOH ; 

 and 0.2 ml. of 40% aqueous urea solu- 

 tion. The diluted a-naphthol is pre- 

 pared at the moment of use by diluting 

 a stock solution (1% crystallized n- 

 naphthol in 96% alcohol) 1 : 10 with 40% 

 alcohol. The watch glass containing 

 the liquids is placed in an ice-bath and 

 the temperature of the reaction fluid 

 inside it must be 0.5°C. 



"3. After 12-15 minutes add 0.2 ml. 

 of a 2% solution of NaOBr. This re- 

 agent is allowed to act for 3 minutes and 

 the solution must be well stirred during 

 this time. The 2% NaOBr must be 

 freshlj' prepared by pouring 2 g. (or 

 approximately 0.7 ml.) of liquid bro- 

 mine into 100 ml. of 5% NaOH, with 

 agitation and cooling. 



"4. Add another 0.2 ml. of 40% urea 

 solution, stir, and immediately after- 

 ward, 



"5. Add another 0.2 ml. of 2% NaOBr 

 and stir well. The coloration attains 

 its maximum after 3-5 minutes and 

 would last only for a short time if it 

 were not stabilized. To stabilize the 

 coloration: 



"6. Take the pieces out of the reac- 

 tion mixture and immerse in pure 

 glycerin for 2-3 minutes and then trans- 

 fer to fresh glycerin. Repeat the opera- 

 tion another two or three times. The 

 passage through 4 glycerin baths is 

 sufficient to stabilize the coloration 

 for some months, even if the pieces are 

 left at room temperature. (We have 

 not mentioned this improvement in 

 any previous publication.) 



"Besides this procedure, which we 

 may call the normal method, there is 

 also another method which results in 

 stronger colorations and very satisfac- 

 tory preparations. To accomplish this, 

 after step 5 the pieces are taken off the 

 reaction liquid and immersed in NaOBr 

 solution for not more than 3 minutes. 

 Afterwards the coloration is stabilized 

 in glycerin, as in the normal procedure. 

 The pieces are mounted and observed 

 in pure glycerin. 



"This reaction is specific for guani- 

 dine derivatives in which only one H- 

 atom of one amino group is substituted 

 by a radical of the alkyl or fatty acid 

 type. In proteic compounds it is 

 specific for arginine. As all proteins 

 hitherto analyzed possess arginine in 

 their molecules, the reaction may be 

 used to demonstrate the presence of 

 proteins in general, other compounds 

 with a reactive guanidine group being 

 rare. The test may also be used to 

 characterize the basic proteins." 

 Arginine Reaction. The method of Thomas, 

 Lloyd E., Stain Techn., 1950, 25, 143-148, 

 has been modified as follows (unpub- 

 lished) written byL. E. Thomas, Dept. 

 of Biochemistry, University of Missouri, 

 Columbia. July 8, 1951 — Bouin's fixa- 

 tive has given the best results. Formal- 

 dehyde (4%) is almost as good. Carnoy's 

 (6:3:1) may be used. After fixation pre- 

 pare paraffin sections. 



1. Remove the paraffin from the slide 

 with xylene and pass it through the 

 alcohols to 70%. 



2. Place the slide in a 0.3% aq. of 

 8-hydroxyquinoline (oxine) in a Coplin 

 jar for 15 min. at room temperature. 

 This solution is prepared by diluting 

 with water a 1.0% stock solution of 

 oxine in absolute alcohol. 



3. Move the slide with a quick motion 

 {not allowing it to drain) into a 0.15 N 

 sodium hypochlorite solution (0.015 N 

 with respect to potassium hydro.xide). 

 This solution is prepared by standardiz- 



