INTRODUCTION AND METHODS 



of dark-adapted human eyes. One of the extracts, prepared from an 

 unwashed retina, was heavily contaminated with retinal blood. The 

 density spectra consequently show the a-, p- and y-bands of oxy- 

 haemoglobin. The other extract, prepared from a retina which had 

 been washed in several changes of acid buffer, was free from blood, 

 and contained only a relatively small proportion of yellow impurities. 

 For both extracts, however, the difference spectra (lower part of 

 Fig. 1.10), are in close agreement. 



The difference spectrum for human visual purple shows that, on 

 bleaching, density is lost at all wavelengths longer than 425 m^, and 

 maximally at about 500 mju. Below 425 mju the density increases on 

 bleaching owing to the formation of a product with greater absorp- 

 tion than the parent pigment. At 425 mju, the isosbestic point, the 

 density is unchanged by bleaching. 



For the difference spectrum to have a precise significance, it is 

 necessary that the unbleached and bleached states of the solution 

 shall be stable. This requirement imposes some limitations on the 

 experimental conditions (temperature and pH). Control of the pH 

 is also important for the reason that the density spectrum of the 

 product of bleaching depends on the acidity of the solution. Con- 

 venient working conditions are pH, 8-9, and temperature, 20°C. 



Even if it were easy to prepare absolutely pure solutions of the 

 visual pigments, the difference spectrum would still be aa indis- 

 pensable analytic function. In the examples shown in Fig. 1.10, the 

 solutions were bleached to completion by a single exposure to Hght. 

 But more information can be obtained when solutions are bleached 

 by a series of exposures. Each instalment of bleaching may be 

 represented as a difference spectrum, and hence compared with any 

 other. In this way the homogeneity of the solution can be tested 

 (see Chapter 6). 



REFERENCES 



Bridges, C. D. B. (1955). The physical chemistry of visual pigments. Ph.D. 



Thesis, London. 

 Collins, F. D., Lxdve, R. M. and Morton, R. A. (1952). Studies in 



rhodopsin. 4. Preparation of rhodopsin. Biochem. /., 51, 292-298. 

 Crawford, B. H. (1949). The scotopic visibility function. Proc. phys. 



Soc. B, 62, 321-334. 

 Crescitelli, F. and Dartnall, H. J. A. (1953). Human visual purple. 



Nature, Lond., 112, 195-196. 

 Dartnall, H. J. A. (1952). Visual pigment 467, a photosensitive pigment 



present in tench retinae. /. Physiol., 116, 257-289. 



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