(I. WEBER 



103 



ihc clinicthylainino group becomes a proton acceptor in a hydrogen 

 bond with the water which has jiresently come into contact with it. 

 Fhiorescence excitation sjjectra of the DNS emission show also the 

 transfer of electronic energy from the tryptophan residues to the DNS 

 and the disappearance of the transfer after twenty hours' digestion 

 of the protein with chymotrypsin, which by l^reaking up the mole- 

 cide into a series of independent peptides causes the tryptophan and 

 the naphthalene sidlonamide to be widely separated from each other 

 (Fig. 17) . A recent appHcation of this energy transfer method to the 

 determination of protein expansion is shown in Fig. 18, where the ac- 

 tion of methyl-ethyl carbinol at 3°C and 38°C is shown. The physical 



FLUORESCENCE EXCITATION SPECTRA 

 OF BCVINE SERUM ALBUMIN CONJUGATED 

 WITH DNS 



l-Sgroups DNS/molecule 



300- 



c 



3 

 la 



u 



8 



t. 



o 



D 



200- 



) Native 



• Digested for 20hrs. 

 with Chymotrypsin 



100 



240 



280 



320 360 



X(mu) 



400 



Fig. 17. The eHect of digestion with thymotiypsiii upon the Huorescence excitation 

 spectrum of bovine serum albumin conjugated with dimethylamino napthalene 

 .sulfochloride (D\S). 



separation of parts of the protein produced by the penetration of the 

 carbinol is followed by the disappearance of the energy transfer. 

 These effects can be analyzed quantitatively by measurements of the 

 protein (tryptophan) fluorescence respectively in the native species 

 and in the DNS conjugate. If the two fluorescence efficiencies are 

 respectively F^ and F^, application of the simple transfer theory re- 

 cently described (28) gives 



Fr 



- 1 = (R'rY 



where R is the characteristic distance for the transfer from trytophan 

 to DNS and r an effective average distance which separates both these 



