Studies of the reaction between proteins and reducing sugars in the ‘dry’ state II. Further observations on the formation of the casein-glucose complex

Abstract

1. 1. The reaction of free amino groups of casein with glucose has been furher investigated by Van Slyke determinations. 1.1. a) The extent of the reaction at a water content corresponding to 20% R.H. has been found to depend on the degree of hydration of the protein surface. Most of the individual amino groups appear to have the same reactivity. 1.2. b)At 70% R.H. rate and extent of the reaction approach a maximum when three equivalents of glucose are present, this quantity approximating to that required to provide a unimolecular layer on the surface of the protein. 2. 2. 1.2.4 fluorodinitrobenzene has also been used for estimation of the free amino groups. 1. a)It has been shown that all the ϵ-amino groups of the lysine side chains (51 mg amino-N/g T.N.) are free to react with the reagent, and that a small quantity of free α-amino groups ( ca i mg amino-N/g T.N.) occur at the ends of polypeptide chains: these α-groups are derived from at least five amino acids, one of which is lysine. 1. b) The loss of amino-N during the reaction with glucose, as found by the Van Slyke method, has been quantitatively confirmed. Both the ϵ-amino groups and the α-amino groups have approximately the same reactivity towards glucose. 1. 3. The rate of disapperance and the fate of the glucose have been followed by various procedures, including use of the specific glucose oxidase (notatin). 1. a) Combination of the free amino groups of casein, each with one molecule of glucose, has been confirmed as the most rapid and virtually the only reaction occurring at 20% R.H., and, in the earliest stages at 70% R.H. As storage proceeds at the higher humidity progressively more carbohydrate becomes bound to the protein than can be accounted for by combination with free amino groups: its weight indicates an average loss of one molecule of water per glucose molecule. 2. b) The nature of the union between the extra carbohydrate and the protein is still uncertain. It may be a chemical union with reactive groups of the protein molecule other than amino groups, i.e., with amino acid residues other than those of lysine. Alternatively, but less probably, it may be the result of caramelization of the glucose followed by adsorption of the product on to the protein. 3. c) None of the carbohydrate combined with (or adsorbed by) the casein can be oxidized by notatin. 4. d) Hydrolysis with dilute acid or alkali fails to regenerate any glucose (or fructose) from the casein-glucose complex, although other reducing substances including furfurals are liberated by the acid.