THE VISUAL PIGMENTS 



doing this is by the use of formaldehyde which reacts with amino 

 groups as follows : 



R— NH3 z^ R— NH2 + H+ 



R— NH2 + HCHO ^ R— NHCCHgOH) 



R— NHCCHgOH) + HCHO <± R— N(CH20H)2 



Since these reactions are all reversible it is not possible to ensure that 

 all free amino groups are blocked. A state of equilibrium will be set 

 up in which a proportion (depending on conditions) will be blocked 

 and the remainder will be free. 



To ensure that in a visual purple solution to which formaldehyde 

 had been added the proportion of the free amino groups was not 

 underestimated Collins assumed that all amino groups were in the 

 form of tyrosine (the amino acid with the least affinity for formalde- 

 hyde). According to levy and silberman (1937), if a solution of 



tyrosine is made 10 molar in formaldehyde and the pH adjusted to 9, 



+ 

 the proportion of free tyrosine (R — NH3 or R — NHg) to combined 



(R— NH(CH20H) or R— N(CH20H)2) is 0-0036. 



Solutions of visual purple which were 13 x 10~^ molar in respect 

 of retinene were found to contain 3 micrograms of amino-nitrogen 

 per milHhtre. To be on the safe side, Collins assumed an upper 

 limit of 5 micrograms per millilitre which is equivalent to 360 X 10~® 

 molar. If now, to such a solution, formaldehyde is added so that 

 the final mixture is 10 molar in formaldehyde, and the pH is adjusted 

 to 9, then the concentration of free amino groups when equihbrium 

 is estabhshed will be 0-0036 x 360 x 10-^ molar = 1-3 x lO-^ 

 molar. Since the potential concentration of retinene in the bleached 

 solution is 13 X 10~^ molar this means that at most, only 10 per cent 

 can combine with protein to form indicator yellow. On the other 

 hand, if the C — N Unk in visual purple is unbroken on bleaching, 

 then the previous blocking of free amino-groups will not affect the 

 amount of indicator yellow, which, accordingly, should approach 

 100 per cent. 



There is, however, an additional complication. If retinene methy- 

 limine (an analogue of indicator yellow) is dissolved in water with 

 the aid of a 'solubilizer,' and the pH adjusted to 9-3, the absorption 

 maximum changes from 363 m// (cf. alkahne indicator yellow) to 

 380 m^ (free retinene). This hydrolysis is a monomolecular reaction 



112 



