2i6 Journal of Agricultural Research voi. in. No. 3 



hemoglobin. In pure solutions nitric oxid is insoluble in ether. The 

 nature of this ether-soluble derivative of NO-hemoglobin will be dis- 

 cussed in connection with the color of cooked salted meats. 



Preparation of pure NO-hemoglobin in dry condition. — Twenty- 

 five c. c. of a concentrated solution of NO-hemoglobin were cooled to 

 0° C, 6 c. c. of absolute alcohol previously cooled to the same tem- 

 perature were added, and the dish was gently rotated. An abundant 

 quantity of dark cherry-red crystals formed immediately. The dish 

 was covered, placed in a refrigerated compartment for 24 hours at a 

 temperature of —4° C, and the contents then filtered with the aid of 

 suction in a room held at a temperature of -1- 1° to -1-4° C. There was 

 obtained a quantity of reddish brown crystals which were partly soluble 

 in water, giving a reddish brown solution which showed a spectrum of 

 NO-hemoglobin contaminated by the presence of methemoglobin. The 

 material was not sufficiently soluble in water to allow of recrystalliza- 

 tion. 



A large number of trials were made with various methods of procedure 

 in an endeavor to obtain pure NO-hemoglobin in dry condition, but 

 without much success. CrystalHzation by a method using alcohol 

 seems to change the NO-hemoglobin, in part at least, to methemoglobin. 



When crystallization was carried on in the presence of a reducing 

 agent — e. g., hydrazin hydrate or Stokes's solution — moist crystals could 

 be obtained which showed a spectrum of NO-hemoglobin free from 

 methemoglobin; but on drying the crystals in vacuo over sulphuric 

 acid a change to methemoglobin took place. 



Crystallization of NO-hemoglobin. — In general, the method used 

 by Reichert and Brown (1909) for the crystallization of hemoglobin and 

 oxyhemoglobin was followed. In brief, the procedure was as follows: 

 A pure concentrated solution of NO-hemoglobin was prepared by the 

 methods previously described, and was examined spectroscopically to 

 determine its freedom from other hemoglobin derivatives. Ammonium 

 oxalate, in the proportion of 2 gm. to 100 c. c, was then added to the 

 solution, which was then shaken to dissolve the salt. All subsequent 

 procedure was carried on in a refrigerated room at a temperature of -t- 1° 

 to+5°C. 



A few drops of the NO-hemoglobin solution were placed on a micro- 

 scopic slide and allowed to evaporate until a heavy, dry proteid ring 

 had formed around the drop. The cover glass was then carefully applied 

 so as to exclude air bubbles, and the edges were sealed with balsam. 

 Microscopic examination was made immediately after mounting and at 

 intervals thereafter, according to the rate of crystal formation. The 

 formation of crystals started at the dried proteid ring and proceeded 

 toward the center of the mount, although occasionally crystals of ammo- 

 nium oxalate would form, and the hemoglobin crystals would start 

 from this base. 



