180 J. E. O'Hagan 



electrons. This means that neither the pyrrole nitrogens nor the metal atom 

 are capable of further combination, only reactive side-chains can effect 

 attachment to other compounds. The covalent linkage of the metal resembles, 

 too, that of the iron in oxy- and carboxyhaemoglobins, and the resonance 

 state of these metalloporphyrins might be expected to be akin to that of the 

 haem in those proteins. 



In the studies reported here, nickel mesoporphyrin was employed, both 

 because it was found to be more readily prepared in pure form than the 

 corresponding protoporphyrin complex and because its use eliminated con- 

 fusion in interpretation of results, through possible (though unhkely) linkage 

 to protein through vinyl groups. Hill and Holden (1926) had shown that it 

 combined with ox apohaemoglobin, as also did nickel protoporphyrin 

 (Holden, 1941). 



To investigate attachment, 10 ml of buffer was placed into each of a series 

 of tubes, then 0-1 ml 5x 10"*M apohaemoglobin solution (assumed 

 M.W. = 16,500) and 0-1 ml 1 x 10^^ m nickel mesoporphyrin (1-34 mg 

 pigment + 2 ml 0-05 n NaOH + 8 ml water) added. Other series replacing 

 the apohaemoglobin solution or nickel mesoporphyrin solution with water 

 were prepared at the same time and the tubes containing the three series 

 stood at 1° for 16 hr and then at 21° for 2 hr before reading absorbances at 

 389 mfi, using a cuvette of 40 mm path length. For the apomyoglobin 

 experiments the undiluted protein solution used was 2 x 10~^ m and the 

 absorbances read at 410 mfi. Excess apoprotein was used because of the 

 likehhood of a small variable quantity of denatured protein being present 

 (O'Hagan, 1960), not removable by any treatment yet described, and likely 

 to precipitate on bringing solutions to room temperature. 



In order to rule out 'protective colloid' effects or non-specific attachment, 

 carboxyhaemoglobin and ferrimyoglobin were substituted for the apoproteins 

 in the experiments. A small peak centred about pH 9 was found with the 

 haemoglobin, while no attachment was detected with the myoglobin. The 

 results, shown in Fig. 3, indicate that attachment to the apohaemoglobin 

 occurs over the pH range 5-12 with two maxima at about pH 7-4 and 10-0. 

 At pH 9-8 nickel mesoporphyrin had an absorption peak at 380 m/;, intensi- 

 fying and moving to 389 m/t (pH 5-7, 8-0, 9-95) on addition of apohaemoglobin 

 (cf. with caffeine, 392-5 m/u). With apomyoglobin the stability range was 

 wider and the peak of the absorption curve shifted much further to 410 m/<. 

 A difference in the position of the peaks with apohaemoglobin and apomyo- 

 globin is in accord with the finding of differences by Hill (1939) when proto- 

 porphyrin was added to these proteins. 



The section of the curve with maximum centred at about pH 7-4 for the 

 apohaemoglobin complex is strongly suggestive of imidazolium combination 

 with one or both of the propionates. The other section with maximum at 

 about pH 10-0 varied in height and width with the preparation and is most 



