The Haem-Globin Linkage 1 75 



in crystal structure in terms of such linkages. They have therefore been 

 examined with this in view and, as a result, a new interpretation of the 

 structural and functional relationships of haemoglobins and myoglobins is 

 presented. 



MATERIALS AND METHODS 

 Aetiohaemin III 

 The preparation was as described by O'Hagan (1960). 



Nickel Mesoporphyrin IX 



Mesoporphyrin IX was prepared by the method of Grinstein and Watson 

 (1943) from dimethylprotoporphyrin IX (Grinstein, 1947). In 5% w/v HCl 

 the absorption spectrum had bands at I, 590-7; la, 570-5; II, 547-5 m// 

 (order of intensity II > I > la, with the Hartridge Reversion Spectroscope). 

 Bands I and la were 1 m/i lower than those reported by Fischer and Orth 

 (1937). Two methods of conversion to the nickel complex were employed. 

 The first followed the technique used by Fischer and Piitzer (1926) to prepare 

 nickel protoporphyrin, but the yield of crystals, even after standing several 

 days in the refrigerator, was poor. The second, a very simple method of 

 preparation, was as follows. About 0-1 g mesoporphyrin was dissolved in 

 about 5 ml acetic acid, the solution was quickly heated to boiling and nickel 

 acetate in acetic acid (prepared by leaving a piece of pure nickel, just covered 

 with acetic acid, in a beaker for a few days) added drop by drop, with 

 continued boiling, until the fluorescence of the porphyrin under ultra-violet 

 light had disappeared. After cooling and adding \ vol. distilled water, the 

 precipitated nickel mesoporphyrin was centrifuged off, washed with alcohol 

 and ether and dried in a vacuum desiccator over NaOH. It was then dissolved 

 in 0-04 N NaOH, the solution centrifuged to remove a trace of undissolved 

 material, the pigment in the supernatant precipitated with 0-2 N HCl, the 

 precipitate centrifuged off, washed several times with distilled water and dried 

 in a vacuum desiccator over NaOH. The nickel mesoporphyrin prepared by 

 both methods had absorption peaks (Hartridge Reversion Spectroscope) as 

 follows: in dioxane, I, 550-5; II, 513 m/< (order of intensity I> II, cf. 

 Lemberg and Legge, 1949); in pyridine, I, 552-0; II, 514 m/* (order I > II). 



Proteins 



The horse apohaemoglobin was prepared by the method previously 

 described for the human material (O'Hagan, 1960) and the apomyoglobin 

 as reported by O'Hagan and George (1959), and both were estimated spectro- 

 photometrically at 280 m// using e^^ = 13 as calculated by Hanania (1953). 

 Before use, the solutions were left for several days at 1°C after adjustment to 

 pH 7-8 to remove as much denatured material as possible. The 25 % human 

 serum albumin was a special batch of salt-poor albumin for transfusion 



