THE INDIVIDUAL GROUPS OF PROTEINS 477 



at the point of contact and diffuses into the clear zone above it. The 

 blue colour ultimately disappears. The blood should not be left in 

 contact with the peroxide for even as long as 5 minutes before adding 

 the guaiaconic acid solution. 



More usually a few c.c. of I per cent, guaiaconic acid in equal parts 

 of alcohol and water are mixed with half the volume of 3 per cent, 

 hydrogen peroxide and the blood solution added. The blue colour 

 develops and disappears. 



This reaction succeeds when blood is diluted I in 10,000. It is not 

 due to any enzyme as it is given by boiled solutions of blood. It de- 

 pends upon the presence of iron as it is not given by haematoporphyrin 

 but is given by all the derivatives of haemoglobin which contain iron* 

 It may be termed a pseudo-peroxidase reaction (Buckmaster). 



() With Benzidine. 



3 or 4 milligrams of benzidine are dissolved in 2 c.c. of glacial 

 acetic acid; 10 drops are put into a clean test tube and mixed with 

 30 c.c. of 3 per cent, hydrogen peroxide. No colour should appear. 

 A blue-green colour is formed on adding a dilute solution of blood. 

 This reaction has a delicacy of at least I in 100,000 depending on the 

 method of carrying out the reaction. 



(c) With A loin. 



25 gm. of aloin are dissolved in 25 c.c. of water + 25 c.c. of alcohol; 

 10-20 drops of this clear yellow solution are mixed with twice the volume of 

 3 per cent, hydrogen peroxide and the blood solution is added. The colour 

 becomes a purple red. 



This reaction is less sensitive than the guaiacum reaction. 



(d) With Malachite Green. 



25 gm. of the leucobase of malachite green are dissolved in 12 c.c of 

 glacial acetic acid + 163 c.c. of water. 



10 drops of this solution are mixed with 3 volumes of 3 per cent, 

 hydrogen peroxide solution and the solution of blood, previously boiled, is 

 added. The colour becomes green. This reaction shows i part of blood in 

 100,000. 



(e) With p-Phenylenediamine Hydrochloride. 



A -5 per cent, solution of this substance in water is prepared. 



1-2 drops are mixed with 15-20 drops of 3 per cent, hydrogen 

 peroxide solution and the blood solution is added. An olive-green colour is 

 slowly formed. If a few drops of dilute acetic acid be added to the mixture, 

 the green colour changes to brown- red. 



(/) With Phenolphthalin. 



032 gm. of phenolphthalin are dissolved in 21 c.c. of 'iN pure sodium 

 hydroxide and diluted to 100 c.c. with water, -i c.c. of N hydrogen peroxide 

 is added. On the addition of blood solution, the phenolphthalin is oxidised 

 to phenolphthalein with the formation of a red colour. 



This reaction is sensitive to i part of blood in 1,000,000. 



