The Isolation, Purification and Properties of Haemin a 327 



when its ethereal solution is washed with neutral sodium chloride solution. 

 In contrast to protohaematin, haematin a does not react, or reacts very little, 

 with cyanide in alkaline solution (0-1 N NaOH). The absorption spectrum is 

 hardly altered by the addition of 0-1 M cyanide. The hydroxyl ion also 

 competes with pyridine for the fifth and sixth co-ordination position of 

 haematin a. Even at pH 9 a solution of haematin a in aqueous pyridine 

 (20% v/v) gives mainly the spectrum of alkaUne haematin a. In aqueous 

 solution, however, without added buffer or alkali, pyridine of this concentra- 

 tion almost completely combines with haematin a to give the ferrihaemo- 

 chrome. With 4-methylimidazole ferrihaemochrome formation is complete 

 with 2% base in weak alkali. Haematin a has thus, like protohaematin, a 

 much greater affinity for the imidazole than for pyridine. 



Ferrous Compounds 



The a-bands of the ferrous haem a compounds with bases or proteins 

 can be grouped in three regions: (1) 587 m/t (pyridine haemochrome), 

 (2) 590 vcifi (serum albumins) and (3) 594-596 m/t (4-methylimidazole haemo- 

 chrome and compounds with native globin and apoperoxidase). With the 

 exception of haem a itself, no compound with a-bands similar to those of 

 cytochromes a + a^ (603-605 m,a) has been found. Similarly ferrous 

 chlorocruorin has an absorption band at 604 mj^i, while that of the pyridine 

 haemochrome hes at 582 m// (Fox, 1924). The serum albumin compounds 

 have a-bands similar to that of cytochrome a^. While the absorption spectra 

 of the pyridine and methylimidazole protohaemochromes differ by no more 

 than about 1 mix, those of the corresponding haemochromes a differ by 

 7 m/<. According to the position of the a-band the haem a iron may be bound 

 to the imidazole of both globin and apoperoxidase, whereas it is assumed that 

 in horseradish peroxidase the protohaem iron is bound to a carboxyl group 

 of the protein. The considerable differences between the spectra of ferrous 

 protohaemoglobin, protohaemperoxidase and protohaeraalbumin are not 

 observed in the series of haem a compounds, nor does the compound 

 with human serum albumin differ in its spectrum from that of ox serum 

 albumin. 



The absorption maxima of the a-bands of alkali-denatured protein haemo- 

 chromes (but not of urea-denatured globin haemochrome in neutral solution) 

 were found at 573-575 m/<, 12-14 m/< to the blue compared to the band of 

 pyridine haemochrome. This difference is surprising in view of the fact that 

 with protohaem denatured proteins give haemochromes with a-bands within 

 1-2 m/i of 558 m/^. Possibly the denatured proteins react with the aldehyde 

 group in alkaUne solution; this reaction also appears to occur with chloro- 

 cruorin, whose absorption band of 604 m/< is shifted to 569 m// on 

 denaturation by alkali (Fox, 1924). 



The millimolar extinctions of the a-bands {e^^ vary between 29-30 for 



