214 Journal of Agricultural Research voi. m. N0.3 



bound oxygen of the oxyhemoglobin with the nitric oxid to form nitrogen 

 peroxid, which, as has just been noted, acts upon the hemoglobin to form 

 methemoglobin. 



Some of the earlier investigators have found that small quantities of 

 oxyhemoglobin could be reduced to hemoglobin by means of hydrogen; 

 but it has been the experience of the writer that, when working with any 

 considerable quantity of oxyhemoglobin, practically no reduction took 

 place even after passing a current of hydrogen through an oxyhemoglobin 

 solution for several hours. In practice it was found best to reduce 

 oxyhemoglobin to hemoglobin by means of hydrazin hydrate before 

 saturation with nitric oxid. 



Properties. — NO-hemoglobin in concentrated solution has a dark 

 cherry-red color; in dilute solutions it has a light cherry-red color, in 

 contrast to the bright-red color of oxyhemoglobin or to the purple-red 

 color of hemoglobin in solutions of the same concentration. In solutions 

 free from methemoglobin NO-hemoglobin is quite stable, and solutions 

 of the compound have been kept in a refrigerated room at 32° to 35° F. 

 for several weeks without apparent change. On boiHng a solution of 

 NO-hemoglobin a brick-red precipitate is formed, in contrast to the 

 dark-brown precipitate formed on boiling a solution of oxyhemoglobin 

 or hemoglobin. 



NO-hemoglobin shows a characteristic spectrum consisting of a heavy 

 band just at the right of the D line and a somewhat lighter and wider 

 band a trifle to the left of the E line (fig. i). These absorption bands 

 occupy practically the same positions as those of oxyhemoglobin, but 

 are distinguishable from the latter in solutions of the same concentration 

 by lower intensity, less sharply defined edges, and by the fact that when 

 a solution of oxyhemoglobin is treated with a reducing agent — e. g., 

 hydrazin hydrate — the characteristic single broad band of hemoglobin 

 appears, while on treating a solution of NO-hemoglobin with the same 

 reagent, no reduction takes place and the bands are not affected. 



NO-hemoglobin is practically unaffected on treatment with potassium 

 ferricyanid in neutral solution, with vStokes's solution (ammoniacal ferro- 

 tartrate), with sodium nitrite, or with hydrazin hydrate, but is grad- 

 ually reduced by potassium ferricyanid in acid solution. 



When a solution of NO-hemoglobin is treated with ether in the pres- 

 ence of a small quantity of alcohol or of dilute acid, a bright-red colored 

 extract is obtained which shows a distinct absorption band just at the 

 right of the D Une, and occasionally, in concentrated solution, a faint 

 band at the left of the E line. In the absence of acid or alcohol no 

 color is extracted by ether. In a previous paper the writer considered 

 that the color extracted under the above conditions was NO-hemoglobin, 

 but from work which he has done since that time it is evident that the 

 color extracted by ether is a derivative of NO-hemoglobin, produced 

 apparently by the reducing action of the alcohol or acid upon the NO- 



