Formol titration method

The ninhydrin reaction (see Basic Protocol 1), the TNBS reaction (see Alternate Protocol 1), the fluorescamine reaction (see Alternate Protocol 2), and formol titration (see Alternate Protocol 3) all evaluate released amino groups by comparing the amounts of free amino groups before and after hydrolysis. The first three methods are spectro-photometric techniques, whereas the fourth is a potentiometric technique. The first and second are chromogenic techniques, whereas the third is fluorometric. These techniques are usually performed as time-course experiments. As the hydrolysis reaction proceeds, aliquots (samples) of the reaction are taken periodically and treated with a test reagent. Products of this reaction are proportional to the amount of free amino groups at each time point. 

Formol titration is a method that estimates amino groups by titration with NaOFI and a phenolphthalein indicator (Vakaleris et ah, 1960). Addition of formaldehyde to the neutralized mixture reduces the pFI by making the amino groups less basic. The amount of NaOFI required to retitrate the mixture has been used as an indicator of proteolysis. Another titrimetric method relies on the increase in buffering capacity of the cheese during ripening and has been applied to study proteolysis in Swiss cheese (Lucey et ah, 1993). 

Formol titration and other methods of determining terminal amino groups are also used to measure the average molecular weight of protein hydrolysates, but they are imprecise because —NH2 groups of amino acid side chains also react and the molecular masses are usually underestimated. 

Another kind of titration after transformation is provided by the formol titration of a-amino acids (Sprensen s method). We have already seen that it is impossible to titrate them in water with sodium hydroxide, because of their macroscopic ionization constant on the order of 10 -. The reaction with the hydroxide ion is not sufficiently quantitative to be used as a titration reaction ... 

---