98 LIGHT AND LIFE 



fluorescence is quenched remains obscure, some important factors 

 have been exckided: 



(1) Methylation of the OH groups of tyrosine with dimethyl sul- 

 fate fails to restore the tyrosine fluorescence. Thus transfer 

 of the H of the OH group to a hydrogen-bonding structure 

 to yield transitorily the non-fluorescent phenolate may be 

 excluded. 



(2) Reduction of the S-S groups with sodium borohydride followed 

 by further stabilization with iodoacetate to give the S-car- 

 boxymethyl derivative does not change appreciably the fluores- 

 cence of the protein (Churchich, impublished) . 



The changes in quantum yield of several proteins with pW have 

 been studied by White (31). Particularly interesting is the com- 

 parison of bovine serum albumin, containing two tryptophan resi- 

 dues in the molecule, with human serum albumin, containing only 

 one. In the latter, the quenching by H ions is half completed at 

 pW 0.5, while in the former the pW fluorescence curve has two 

 distinct steps, with midpoints at pH 3.5 and pH 0.5. Comparison with 

 free tryptophan and its derivatives is immediate. Of the two residues 

 of bovine albumin one must be located in the vicinity of a COOH 

 group of aspartic or glutamic acid, while the other, as well as the 

 unique residue in human albumin, must be located away from any 

 such groups, so that they are quenched by the protons in the medium 

 in much tiie same manner as the derivatives of tryptophan in which 

 the COOH group is chemically engaged. Steps on the acid side of 

 the jbH fluorescence curve are also found in lysozyme, which shows 

 steps on the alkaline side as well. These data indicate differences in 

 the accessibility of the different tryptophan residues to protons and 

 are difficult to reconcile with any theory which envisages the free 

 migration of protons over the protein surface. 



Fluorescence Lifetime and Polarization 

 An estimation of the lifetimes of the excited states in tryptophan 

 in the proteins by methods already discussed gives values of 1-3 

 ni/xsec. Since the rotational relaxation times of most globular pro- 

 teins are 20 to 100 times longer (13, 25), a conspicious change in 

 polarization with temperature or viscosity may not be expected. 

 Polarization measurements should be useful in the determination 

 of the rotational relaxation times of protein fragments of up to a 

 few thousand in molecular weight. 



The polarization spectrum of the fluorescence of proteins of Class B 



