MODIFIED REACTIVITY OF HAEMOGLOBIN FOLLOWING LIGHT ABSORPTION BY Q. GIBSON Sheffield University Received 16th January, 1959 When carboxyhaemoglobin is broken down by the action of light, the newly formed reduced haemoglobin has a higher rate of combination with carbon monoxide than ordinary reduced haemoglobin. " Newly-formed " haemoglobin is converted to " ordin- ary " haemoglobin with a rate constant of about 200 sec-1 at pH 9 and 0;O. The rate constant for the combination of "newly-formed" haemoglobin with CO is 1-8 x 106 M-1 sec-1 at 0;O and the activation energy is 5.6 kcal (cf. ordinary haemoglobin 4 x 104 M-1 sec-1 and 10.5 kcal). In 1896 Haldane and Lorrain-Smith 1 found that the results of the carmine titration method for determining the composition of mixtures of oxy- and carboxy- haemoglobin depended on the lighting of the laboratory, and went on to prove that carboxyhaemoglobin (COHb) is photosensitive.It has since been shown by Buecher and Kaspers 2 that the absorption and action spectra of carboxymyo- globin are identical over the range 200-450 mp ; thus energy transfer must take place from the aromatic amino-acids of the protein to the haem group. The quantum yield was 1 throughout, so this transfer is clearly an efficient process. It has been known for many years that light of wavelengths shorter than 300 mp will produce major irreversible changes in proteins, and haemoglobin is among those attacked. With visible or near ultra-violet light, which is absorbed chiefly by the haem group, it has been supposed, however, that the dissociation COHb +- CO + Hb is the only reaction taking place.Experiments described by Gibson 3 suggest that this may not be true, as under some circumstances the immediate product of the photodecomposition of COHb is not ordinary reduced haemo- globin (Hb) but a short-lived quickly-reacting species which will be written Hb*. Several explanations of the observations are possible and will be briefly discussed. Gibson 3 found that, following the photochemical dissociation of COHb by light of wavelengths longer than 310 mp, there was present in the solutions a pig- ment whose spectrum in the Soret region was closely similar to, but not identical with, that of reduced haemoglobin. This material Hb* was converted spontan- eously into Hb with a rate constant at 0" and pH 9.1 of 250 sec-1.It reacted with CO to re-form HbCO with a rate constant at 0" of 2 x 106 M-1 sec-1 (cf. the rate constant for the combination of Hb with CO at pH 9.1 and 0" of 5 x 104 M-1 sec-1 given by Gibson and Roughton 4). The energy of activation for Hb* combining with CO was 5.6 kcal at pH 10.6, compared with 10.5 kcal for Hb combining with CO. The proportion of Hb* formed is a function of pH, being least at pH 6-6. It appears to depend also on the rate at which CO is removed from combination by the photolysis flash, though for technical reasons few flash intensities have been examined. These findings may be related to Gibson and Roughton's 4 measurements of the rate of combination of the four successive molecules of CO with Hb. They found that whereas the first three molecules to combine do so at roughly similar rates, the fourth molecule combines about 40 times faster.This difference in behaviour between free and combined Hb is thought to be due to a structural change in the protein rather than to a difference in individual haem molecules; 142Q. GIBSON 143 certainly, saturation is associated with changes in crystal form and solubility, as well as in the isoelectric point. With these results in mind, it seems reasonable to think of Hb* as a haemoglobin molecule which has lost its ligand molecules through photochemical action in a space of time so brief that the reorganization of the protein normally associated with the loss of the second ligand molecule has not had time to occur. In slightly different words, Hb* may be regarded as combined haemoglobin minus its ligand molecules.This qualitative picture will explain satisfactorily the high rate of combination of Hb* with COY which is similar to the rate for the combination of the fourth and last molecule of CO with Hb, i.e. the reaction Hb4(C0)3 + CO +- Hb4(C0)4 : the similarity extends indeed to the ratio between the rates of combination of 0 2 and CO. Thus for Hb* the ratio is 4/1 ; for Hb4(C0)3 Gibson and Roughton 5 found 3.5 : 1. The nature of the change in the protein is not known. It may well involve sulphydryl groups, since the transition Hb* -+ Hb is much slower in the presence of mercurials. St. George and Pauling 6 have suggested that the haem groups may lie in a " crevice " in the protein, and it is naturally tempting to relate the increase in the rate of CO combination on passing from Hb4 to Hb4(C0)3 to the opening up of such a crevice structure.The rates and activation energies for the reactions Hb* + CO and Hb + CO do not support such an idea, for the difference of 5 kcal should correspond to a difference in rate of about 1000-fold, instead of the observed %-fold, while, if steric factors were favourable, a still greater increase might be looked for. In fact, the steric factor is less favourable for the combination of Hb* than for Hb. Finally, it is interesting to consider if the observations should be regarded as an example of energy transfer. A photochemical effect is produced giving rise to Hb*, and this action is produced at a distance since the protein rather than the haem is implicated. On the other hand, unless all the ligand molecules are stripped from the haemoglobin molecule within a brief space of time, Hb* will not appear, as the changes in the protein required to give Hb will take place faster than the removal of ligand. On this picture, the removal of the ligand molecule would be a trigger step followed by a spontaneous change in the protein. Thanks are due to the Medical Research Council for contributions to the cost of the work. 1 Haldane and Lorrain-Smith, J. Physiol., 1896, 20, 497. 2 Buecher and Kaspers, Biochim. Biophys. Acta, 1947, 1, 21. 3 Gibson, Biochem. J., 1959, 71, 293. 4 Gibson and Roughton, Proc. Roy. SOC. B, 1957,146, 206. 5 Gibson and Roughton, Proc. Roy. SOC. By 1955,143,310. 6 St. George and Pauling, Science, 1951,114,629.