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Spectrophotometric Assay Using o-Phthaldialdehyde for Determination of Proteolysis in Milk and Isolated Milk Proteins¹

Department of Food Science, North Carolina State University, Raleigh 27650

ABSTRACT

A rapid, sensitive, and convenient Spectrophotometric assay was developed and characterized for measurement of proteolysis of milk proteins in buffered solutions or in milk. -Amino groups released by hydrolysis react with o-phthaldialdehyde and (ß-mercaptoethanol to form an adduct that absorbs strongly at 340 nm. The absorptivity ( = 6000 M^–1 cm^–1) is similar for all -amino groups. Moreover, the absorptivity of the adduct with both - and -amino groups of proteins is also similar and unaffected by local environment when proteins are denatured in sodium dodecyl sulfate. Thus, background is constant for a particular sample, and -amino groups released by proteolysis can be quantitated accurately. Inclusion of sodium dodecyl sulfate in the assay provides a convenient way to terminate proteolysis and to insure full exposure and complete reaction of amino groups. Because all hydrolytic products are assayed, the method is more accurate than procedures that depend upon properties of aromatic residues (Hull and Lowry methods). Furthermore, the o-phthaldialdehyde Spectrophotometric assay is more rapid and convenient than methods using ninhydrin, 2,4,6-trinitrobenzenesulfonic acid, or fluorescamine. The assay proved to be especially useful for measuring proteolysis in milk from microbial culture organisms such as Streptococcus lactis C2. Because trichloroacetic acid filtrates are used, the method should be adaptable to other dairy products.

¹ Paper No. 8483 of the Journal Series of the North Carolina Agricultural Research Service. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products.

² Dental Research Center and Department of Pathology, University of North Carolina, Dental Research Building 210H, Chapel Hill 27514.

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