Modification of the Secondary Structure of Haemoprotein Molecules 95 



CH3 CHg 



I •/ 



N -N 



HI II 



c c 



\l / \ll / 



Fe Fe 



/w /w 



This conclusion is not entirely unexpected, because we cannot use a linear combination 

 of ■tpj, and v'jb to represent the actual bonding since structures A and B involve diflFerent 

 nuclear positions. Our conclusion that A is energetically favoured is supported by 

 the electron diffraction finding that methyl isocyanide is essentially a linear molecule 

 (Gordy and Pauling, /. Amer. chem. Soc. 64, 2952, 1942). Presumably the binding of 

 ethyl isocyanide by myoglobin and urea-denatured haemoglobin is also essentially 

 represented by structure A. If we adopt the assumption that "back-7r-bonding" is 

 also the main cause of the Bohr effect, we would expect the binding of isocyanide by 

 haem, myoglobin and urea-denatured haemoglobin respectively to be practically 

 independent of pH. 



On the other hand, alkyl isocyanide may be just one of those borderline cases 

 where the energy difference between structures A and B is so small that even additional 

 secondary interaction in the system could reverse the relative stability of these two 

 structures. This suggests an attractive possible explanation of Kaziro and Tsushima's 

 data (this volume, p. 100). If we assume, as illustrated above, that the binding site 

 in native haemoglobin is so crowded with the hydrophobic part of the protein that 

 the isocyanide molecule will have to push the haem and the protein apart somewhat 

 in order to be bonded, then the secondary interactions at the binding site may actually 

 reverse the relative stability of A and B. Because of these secondary interactions, 

 it is conceivable that the overall-AF° for the ethyl isocyanide to be bonded like B 

 above may be larger than that for A, and consequently it exhibits the Bohr effect. 

 The structures near the binding site in myoglobin and urea-denatured haemoglobin 

 respectively are probably much less rigid, and hence the advantage of structure B 

 disappears, so does the Bohr effect. 



Kaziro: I am afraid Wang must have misunderstood my paper. In fact myoglobin 

 shows no Bohr effect with either oxygen or alkylisocyanide. Further, one cannot test 

 whether urea-denatured haemoglobin shows a Bohr effect in Og, because of its great 

 autoxidizability. 



Wang : The existence of a weak Bohr effect in the oxygenation of myoglobin was shown by 

 Theorell {Biochem. Z., 268, 55, 1934). His data are also supported by the results 

 obtained by Chiu and Spencer in my laboratory on the combination of carbon 

 monoxide with myoglobin which showed a definite Bohr effect though not as pro- 

 nounced as in the case of haemoglobin. My remark on the contrasting behaviour of 

 ethyl isocyanide and oxygen respectively toward myoglobin was based on the com- 

 bined information obtained from the present work and Theorell's data. 



Drabkin: I would like to mention that some twenty years ago (in the J. Biol. Chem. and 

 Proc. Soc. Exp. Biol. Med.), I made brief reports both upon the combination of 

 haemin with denatured proteins (haemoglobin, albumin, peptones) with findings 

 similar to the present (and Zeile preceded me in this area), as well as upon the influence 

 of 4 M urea on denaturation with alkali. Haemoglobin in 4 M-urea was found to be 

 denatured 60 times faster than in the absence of urea. Myoglobin, exceedingly stable 

 towards alkali, was denatured about 1000 times faster than in aqueous solution. 

 Urea unfolds the molecule and exposes susceptible groups even in the case of myo- 

 globin, which of course is of lower molecular weight than haemoglobin and contains 

 one, not four, iron atoms per molecule. 



Wang : Williams' work on Fe++-dimethylglyoxime complexes has certainly widened our 

 understanding of the structure of haem and related compounds. But in addition to 



