Chemistry and Biology of Proteins - F. Haurowitz 1953

Hydrolytic Cleavage of Proteins
Determination of Hydrolysis Rate

During Protein Hydrolysis, the Cleavage of peptide bonds proceeds According to the following equation:

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The degree of hydrolytic cleavage of a protein can therefore be assessed by determining the number of newly liberated carboxyl or amino groups, or both simultaneously.

The increase in the number of carboxyl groups can be detected by titration with an alkali in the presence of formaldehyde or ethyl alcohol. Formol titration was first proposed by Sørensen [12], who initially assumed that this reaction yields N-methylene compounds of the following formula: RN = СН₂. Although this assumption proved to be erroneous (see Chapter VII), it should nevertheless be noted that formaldehyde condenses with the amino groups of Amino Acids, causing these groups to lose their basic properties. When amino acids interact with formaldehyde, they most likely form mono- or dimethyl derivatives corresponding to the formula R ∙ NH ∙ СН₂ОН or R ∙ N(CH₂OH)₂ [13, 14]. The number of carboxyl groups in amino acids or Proteins can also be determined by titration in ethyl alcohol [15]. Alcohol titration is made possible by a shift in the pK value of phenolphthalein used as an indicator; whereas the color change of phenolphthalein in aqueous solution occurs near pH 9, in alcoholic solution this change takes place at around pH 12 [16]. At these high pH values, amino acids exhibit acidic properties because the positively charged NH₃ groups are converted into uncharged NН₂ groups, which are unable to neutralize the negatively charged СОО^- groups (see Chapter V).

Since Amino acids are weaker acids than Peptides (see Chapter V, Table 6), peptide hydrolysis is accompanied by a slight decrease in acidity. If carbon dioxide and bicarbonate are present in the medium, peptide hydrolysis will result in the absorption of carbon dioxide, The amount of which can be measured manometrically in a Warburg apparatus [17] by the decrease in carbon dioxide pressure.

The classical METHOD FOR DETERMINING free amino groups liberated during hydrolysis is the Van Slyke method [18]. This method is based on the ability of free amino groups to react with nitrous acid to produce nitrogen gas according to the following equation:

While the d-amino groups of Amino Acids and the terminal d-amino groups of peptides react very rapidly with nitrous acid, the ε-amino groups of Lysine react very slowly. The action of nitrous acid also slowly decomposes ammonia—which is cleaved from the amide groups of asparagine and glutamine during Hydrochloric acid hydrolysis—with The formation of nitrogen [18]. The volume of the resulting nitrogen can be measured either at atmospheric pressure or manometrically under reduced pressure [20]. The Van Slyke method yields excellent results with Most amino acids and peptides. However, it must be taken into account that nitrous acid itself is decomposed by strong reducing agents (such as Cysteine) to yield nitrogen [21]. Determinations of nitrogen in Glycine and its peptides also yield values that exceed the theoretically calculated figures [22, 23].

The number of amino groups released during hydrolysis can also be determined by titrating The amino acid in acetone with hydrochloric acid using naphthol red as an indicator [24], or by titrating amino acids dissolved in glacial acetic acid with perchloric acid using brilliant cresyl blue as an indicator [25].

The course of hydrolysis involves the disappearance of free Water molecules according to the following equation:

The reaction is accompanied by a decrease in volume, which can be measured dilatometrically. This method has been refined to such an extent that it can be used to monitor The rate of Enzymatic hydrolysis in a single drop of the reaction mixture [26].

All the Methods discussed above make it possible to monitor the hydrolytic degradation of proteins by tracking the increase in the number of amino or carboxyl groups liberated during hydrolysis; however, they do not distinguish free amino acids from peptides. Such differentiation is possible by using Reagents that interact simultaneously with both the carboxyl and a-amino groups of the amino acid. The most important reagent of this type is ninhydrin (triketohydrindene hydrate). When aqueous solutions of a-amino acids are heated with ninhydrin, oxidative Cleavage of the amino acids occurs, yielding the corresponding aldehyde, carbon dioxide, and ammonia.

The resulting aldehyde immediately condenses with reduced ninhydrin. The Condensation product has a blue color, the intensity of which can be measured colorimetrically [27]. If heating with ninhydrin is carried out at pH 1–5, gas analysis methods can be used to determine the amount of free amino acids from the amount of carbon dioxide produced. Neither proteins nor peptides react with ninhydrin under these conditions [28].