1042
J. Agric. Food Chem. 1984, 32, 1042-1044
Enzymatic Hydrolysis of Maltosyl and Glucosaminyl Derivatives of P-Lactoglobulin Ralph D. Waniska*l and John E. Kinsella
The in vitro digestibilities of maltosyl and glucosaminyl derivatives of @-lactoglobulin(M-@-LGand G-@-LG,respectively) were measured by using trypsin and a-chymotrypsin. The rate and extent of proteolysis of @-lactoglobulinwere affected by the extent of modification, by the type of carbohydrate residue coupled to @-LG,and by the type of proteolytic enzyme. The rate of hydrolysis increased as the number of residues modified increased. The rates of hydrolysis of M-@-LGderivatives were higher than those of G-@-LGderivatives. This resulted because the tertiary conformation of M-@-LGderivatives was more expanded than that of the modified G-@-LGderivatives. The rates and extents of hydrolysis of M-@-LGand G-@-LGwere greater with a-chymotrypsin than with trypsin because the a-chymotrypsin hydrolyzing sites were increasingly exposed to the surface of the protein as modification increased.
Studies to determine the digestibility and biological value of the chemically modified proteins should be conducted concurrently with research to enhance the functional properties of the proteins. A number of researchers have reported decreased digestibility of chemically modified proteins (Puigserver et al., 1979a,b; Galembeck et al., 1977; Shetty and Kinsella, 1982). Glycosylation of @-lactoglobulin(@-LG)caused significant changes in its chemical and physicochemical properties, e.g., increased viscosity, increased exposure of aromatic amino acid residues to ultraviolet radiation, and decreased a-helical structure of the glycosylated derivatives of @-LG(Waniska and Kinsella, 1984a,b). Since research on the structure and function of proteins should include some measure of the proteins digestibility, the in vitro digestibilities of the glycosylated derivatives of @-LGwere studied. The rate and extent of enzymatic hydrolysis of maltosyl- and glucosaminyl-@-lactoglobulinderivatives (M-@-Lgand G-@-LG,respectively) were determined by using trypsin and a-chymotrypsin. MATERIALS AND METHODS Analytical Methods. Bovine @-lactoglobulin(crystallized and lyophilized; Sigma Chemical Co., St. Louis, MO) was dialyzed against distilled water containing 0.01 % sodium azide and then lyophilized before use. Maltosyl@-lactoglobulin(M-@-LG)derivatives were prepared as reported by Waniska and Kinsella (1984a). The M-@-LG derivatives were prepared by reacting the cyclic carbonate derivative of maltose with @-LGaccording the method of Doane et al. (1967) (Scheme I). Maltose cyclic carbonate was prepared by adding ethyl chloroformate to a solution of maltose and triethylamine in dimethyl sulfoxide. After purification, the activated sugar was reacted with the amino groups of b-LG (16 amino groups per protein) in 0.02 N sodium phosphate buffer (pH 8.0) for 15 h at 22 "C with constant stirring. The derivatized protein was then dialyzed 3 times against 0.10 M sodium chloride and 0.01% sodium azide, and then the protein was dialyzed 3 times against 0.010 M sodium phosphate (pH 6.3) containing 0.01 % sodium azide. This was done to ensure the complete removal of the noncovalently bound carbohydrates. The protein was used immediately or stored at 4 OC until used. The M-0-LG derivatives had 1.3, 2.2,3.5, 5.7,9.1, and 14.5 amino acid residues glycosylated with a total of 1.3, 2.6, 5.7, 9.9, 18.8, and 32.0 maltose groups, respectively Institute of Food Science, Cornel1 University, Ithaca, New York 14853. 'Present address: Cereal Quality Laboratory, Texas A&M University, College Station, TX 77843-2474. 0021-856118411432-1042$01.50/0
Scheme I. Simplified Sequence of Reactions in the Cyclic Carbonate Method Required To Activate Carbohydrates and To Couple the Cyclic Carbonate Derivatives to the Amino Groups of Protein ?-=
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