PREPARATION OF NATIVE GLOBIN 259 



some renatured globin in their preparations. Hill and Holden {1282), 

 however, were the first to isolate native globin from hemoglobin, and 

 to carry out the coupling of hematin to globin under controlled 

 conditions. Their method gave poor yields of native globin. By a 

 modification of the acid acetone procedure, which Hamsik (1119) used 

 for the preparation of hemin, Anson and Mirsky (72) were ultimately 

 able to recover up to 80% native globin from ox hemoglobin, but were 

 less successful with the proteins from other species. The method, or 

 slight modifications of it, remains in general use (625,24-37). 



Washed, laked corpuscles are cooled at 0° C. and added gradually to a 

 tenfold volume of acetone containing 1% cone, hydrochloric acid also cooled 

 to 0° C. The mixture is allowed to stand for two or three minutes and is 

 filtered; the acid-denatured globin is washed with acetone and allowed to 

 dry. All operations are carried out at low temperature. The mixture is then 

 ground in a chilled mortar with water until it dissolves, and carefully titrated 

 with 0.2 A'^ sodium hydroxide until a slight permanent precipitate is formed. 

 After standing for 15 minutes, it is titrated with alkali until the maximum 

 precipitate of denatured protein is formed. The solution of native globin is 

 filtered from the denatured protein after a further thirty minutes (if the 

 neutralization is carried out rapidly, the protein is not renatured). Anson 

 and Mirsky then add ammonium sulfate until the solution is 40% saturated 

 to precipitate any denatured globin remaining in the solution, filter this ofiF, 

 precipitate the native protein by adding additional ammonium sulfate to 

 56% saturation, redissolve, and remove ammonium sulfate by dialysis at 

 low temperature against distilled water. The protein may be further purified 

 by repetition of the salting-out procedure (lli.l8 ,2300) . By freeze-drying, a 

 stable preparation can be obtained. 



Oxyhemoglobin, resynthesized from native globin and alkaline 

 hematin, shows properties slightly different from those of the original 

 hemoglobin (2314). Hill and Holden (1282) observed that the bands 

 of the synthesized compound were shifted a few angstrom units to 

 the blue in hemoglobin, oxyhemoglobin, and carboxyhemoglobin. 

 Investigation of resynthesized carboxyhemoglobin in the ultracentri- 

 fuge showed that its molecular weight was unaltered, but electro- 

 phoretic measurements showed a tendency for the isoelectric point 

 to become slightly more acid (1028). 



Drabkin (625) has prepared denatured globin from crystalline 

 horse myohemoglobin and has succeeded in renaturing it completely 

 and in obtaining 100% yield of synthetic myohemiglobin, which 

 crystallized in the same habit as the original protein. 



