14 INFLUENCE OF TEMPERATURE ON BIOLOGICAL SYSTEMS 



diffusion constants requires rather long times and is subject to uncertain- 

 ties when the protein is not homogeneous. Sedimentation constants do not 

 tell anything unequivocal about the shape of a i)rotein because they de- 

 pend on the molecular weight as well as on the shape; they are dependable 

 indications of unfolding only if an independent measurement of the 

 molecular weight has been made under the same conditions as those pres- 

 ent in the sedimentation run. Flow birefringence is observable with equip- 

 ment that is now available only if the particles are quite long or if the 

 viscosity of the solvent has been raised substantially by adding large 

 amounts of such substances as sucrose or glycerol — which may have im- 

 portant effects of their own on the clenaturation process. Measurements 

 of the dissymmetry of light scattering are not useful in determining par- 

 ticle shapes unless the particle has at least one dimension of the order of 

 1000 A.; this makes the method unsuitable for proteins whose molecular 

 weight is smaller than about 100,000 (which eliminates the method for 

 study of such common proteins as ovalbumin, serum albumin and insulin 

 in their denatured, but unaggregated state) . Low angle x-ray scattering 

 requires apparatus that is not available in most laboratories. Depolariza- 

 tion of fluorescent light requires studies with each protein-dye complex 

 of the lifetime of the excited state of the fluorescent molecule that is used. 

 The measurement of the infra-red absorption of proteins in solutions con- 

 taining water encounters difficulties because of the strong absorption of 

 water over the most readily accessible range of infra-red wave lengths. 

 Except with the chromoproteins such as hemoglobin and cytochrome c, 

 denaturation does not seem to have much effect on the visible and ultra- 

 violet absorption spectrum of proteins. (An important exception is the 

 spectral change that occurs when some proteins are denatured at alkaline 

 pH, which is brought about indirectly by a shift in the dissociation con- 

 stant of the hydroxyl group of tyrosine, refs 3, 31.) Changes with time of 

 the partial specific volume and refractive index are easy to measure at 

 room temperature, but because of the need for very close temperature 

 control, they are not so easy to follow at other temperatures. Furthermore, 

 if the protein unfolds very rapidly on exposure to the denaturing agent, 

 it may be difficult to separate the changes that are caused by denaturation 

 from the changes brought about by the addition of the denaturing agent 

 to the solvent. 



Fortunately two properties, one from each of the two classes mentioned 

 above, are easily measured with apparatus that is either easily constructed 

 or is readily available in most chemical and biochemical laboratories. 

 These two properties are the viscosity and the optical rotation. Largely 

 because of the ease with which they can be measured they arc frequently 



