SIDXEY I'. lElJCK 



127 



>- 



CO 



CO 

 CO 



100 

 80 

 60 

 40 

 ^ 20 



^12 3 4 5 6 7 320 360 400 440 

 APNH C/<molar) EMISSION, mju 



Fig. 13. 1 he emission bands of LDH and of the LDH-(Al'NH),, complex excited 

 at 290 m^. Note the extreme tiuenching of the protein emission band by the 

 coenzyme analogue, and the lelafively strong APNH emission band excited by 

 energy transfer from the protein. The curves on the left show the changes in 

 intensity of protein and nucleotide emission as the protein is titrated with APNH. 

 Note that both curves level off as saturation is approached. Free APNH, excited 

 at 290 nifi, shows negligible emission at 460 m^x on the scale of these curves. 



calculated from the amino acid analysis and the assumption that 

 the tryptojjhan in the jiiotein absorbs like the free amino acid in 

 alkaline solution. The area under the excitation band is about 40 

 per cent of that under the protein absorption curve, a relation which 

 means that about 40 per cent of the light quanta absorbed in the 

 protein are transferred to the coenzyme. On the other hand, the 

 integrated excitation band is roughly 75 per cent of that of the 

 absorption band of the constituent tryptophan. This corresponds 

 approximately to the amount of quenching of the tryptophan emis- 

 sion. The residts are only approximate because we do not know 

 the exact position and shape of the tryptophan absorption band in 

 the protein. The best fit between the observed protein absorption 

 band and the contributions of its constituent tryptophan and tyrosine 

 is obtained with the tryptophan band as shown and two types of 

 tyrosine absorption, 69 per cent corresponding to the phenolic form 

 and 31 per cent to the phenolate ion. The analysis of the curves in 

 Fig. 14 is in accord with the idea (a) that the protein emission is 

 chiefly tryptophan emission, (b) that tryptophan is the donor in 

 the energy transfer to bound coenzyme, and (c) that the amount 



