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

  • Author Footnotes
    2 Dental Research Center and Department of Pathology, University of North Carolina, Dental Research Building 210H, Chapel Hill 27514.
    Frank C. Church
    Footnotes
    2 Dental Research Center and Department of Pathology, University of North Carolina, Dental Research Building 210H, Chapel Hill 27514.
    Affiliations
    Department of Food Science, North Carolina State University, Raleigh 27650
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  • Harold E. Swaisgood
    Affiliations
    Department of Food Science, North Carolina State University, Raleigh 27650
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  • David H. Porter
    Affiliations
    Department of Food Science, North Carolina State University, Raleigh 27650
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  • George L. Catignani
    Affiliations
    Department of Food Science, North Carolina State University, Raleigh 27650
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  • Author Footnotes
    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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      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. α-Amino groups released by hydrolysis react with ο-phthaldialdehyde and (β-mercaptoethanol to form an adduct that absorbs strongly at 340 nm. The absorptivity (ɛ = 6000 M−1 cm−1) is similar for all α-amino groups. Moreover, the absorptivity of the adduct with both α- and ɛ-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 α-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 &ogr;-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.

      References

        • Beeby R.
        The use of fluorescamine at PH 6.0 to follow the action of chymosin on κ-casein and to estimate this protein in milk.
        New Zealand.J. Dairy Sci. Technol. 1980; 15: 99
        • Bensadoun A.
        • Weinstein D.
        Assay of proteins in the presence of interfering materials.
        Anal. Biochem. 1976; 70: 241
        • Benson J.R.
        • Hare P.E.
        o-Phthalaldehyde: Fluorogenic detection of primary amines in the picomole range Comparison with fluorescamine and ninhydrin.
        Proc. Natl. Acad. Sci. USA. 1975; 72: 619
        • Chen R.F.
        • Scott C.
        • Trepman E.
        Fluorescence properties of ophthaldialdehyde derivatives of amino acids.
        Biochim. Biophys. Acta. 1979; 576: 440
        • Chism G.W.
        • Huang A.E.
        • Marschall J.A.
        Sensitive assay for proteases in sterile milk.
        J. Dairy Sci. 1979; 62: 1798
        • Church F.C.
        • Catignani G.L.
        • Swaisgood H.E.
        Hydrolysis of milk proteins by immobilized Streptomyces griseus pronase.
        J. Dairy Sci. 1981; 64: 724
        • Citti J.E.
        • Sandine W.E.
        • Elliker P.R.
        Some observations on the Hull method for measurement of proteolysis in milk.
        J. Dairy Sci. 1963; 46: 337
        • Cliffe A.J.
        • Law B.A.
        A new method for the detection of microbial proteolytic enzymes in milk.
        J. Dairy Res. 1982; 49: 209
        • Exterkate F.A.
        Accumulation of proteinase in the cell wall of Streptococcus cremoris strain AM1 and regulation of its production.
        Arch. Microbiol. 1979; 120: 247
        • Folin O.
        • Ciocalteau V.
        On tyrosine and tryptophane determinations in proteins.
        J. Biol. Chem. 1927; 73: 627
        • Fox P.F.
        Proteinases in dairy technology.
        Netherlands Milk Dairy J. 1981; 35: 233
        • Goodno C.C.
        • Swaisgood H.E.
        • Catignani G.L.
        A fluorimetric assay for available lysine in proteins.
        Anal. Biochem. 1981; 115: 203
        • Hull M.E.
        Studies on milk proteins.II. Colorimetric determination of the partial hydrolysis of the proteins in milk.
        J. Dairy Sci. 1947; 30: 881
        • Law B.A.
        Reviews of the progress of dairy science: Enzymes of psychrotrophic bacteria and their effects on milk and milk products.
        J. Dairy Res. 1979; 46: 573
        • McKay L.L.
        • Baldwin K.A.
        Simultaneous loss of proteinase and lactose-utilizing enzyme activities in Streptococcus lactis and reversal of loss by transduction.
        Appl. Microbiol. 1974; 28: 342
        • Mills O.E.
        • Thomas T.D.
        Release of cell wall-associated proteinase(s) from Lactic streptococci..
        New Zealand.J. Dairy Sci. Technol. 1978; 13: 209
        • Moore S.
        • Stein W.H.
        A modified ninhydrin reagent for the photometric determination of amino acids and related compounds.
        J. Biol. Chem. 1954; 211: 907
        • Nelson J.H.
        Impact of new milk clotting enzymes on cheese technology.
        J. Dairy Sci. 1975; 58: 1739
        • Okuyama T.
        • Satake K.
        On the preparation and properties of 2,4,6- trinitrophenylamino acids and -peptides.
        J. Biochem. 1960; 47: 454
        • Pearce K.N.
        Use of fluorescamine to determine the rate of release of the caseino-macropeptide in rennettreated milk..
        New Zealand.J. Dairy Sci. Technol. 1979; 14: 233
        • Porter D.H.
        • Swaisgood H.E.
        • Catignani G.L.
        A rapid fluorometric assay for measurement of peptidase activity.
        Anal. Biochem. 1982; 123: 41
      1. Reimerdes, E. H., and H. Klostermeyer. 1976. Determination of proteolytic activities on casein substrates. Page 26 in Methods in Enzymology. Vol. XLV. L. Lorand, ed. Academic Press, New York, NY

        • Richardson B.C.
        The purification and characterization of a heat-stable protease from Pseudomonas fluorescent B52.
        New Zealand J. Dairy Sci. Technol. 1981; 16: 195
        • Roth M.
        Fluorescence reaction for amino acids.
        Anal. Chem. 1971; 43: 880
        • Rowlett R.
        • Murphy J.
        A convenient spectrophotometric method for the kinetic analysis of the enzymic hydrolysis of N-acyl peptides using phthaldialdehyde.
        Anal. Biochem. 1981; 112: 163
        • Schwabe C.
        A fluorescent assay for proteolytic enzymes.
        Anal. Biochem. 1973; 53: 484
      2. Segel, I. H. 1975. Ch. 2. Pages 18-99 in Enzyme kinetics. John Wiley and Sons, New York, NY

        • Simons Jr, S.S.
        • Johnson D.F.
        The structure of the fluorescent adduct formed in the reaction of o-phthalaldehyde and thiols with amines.
        J. Am. Chem. Soc. 1976; 98: 7098
        • Simons Jr, S.S.
        • Johnson D.F.
        Reaction of o-phthalaldehyde and thiols with primary amines: Fluorescence properties of 1-alkyl (and aryl) thio-2-alkylisoindoles.
        Anal. Biochem. 1978; 90: 705
        • Svedas V-J.K.
        • Galaev I.J.
        • Borisov I.L.
        • Berezin I.V.
        The interaction of amino acids with ophthaldialdehyde:A kinetic study and spectrophotometric assay of the reaction product.
        Anal. Biochem. 1980; 101: 188
        • Taylor S.
        • Tappel A.L.
        Lysosomal peptidase measurement by sensitive fluorometric amino acid analysis.
        Anal. Biochem. 1973; 56: 140
        • Terzaghi B.E.
        • Sandine W.E.
        Improved medium for lactic streptococci and their bacteriophages.
        Appl. Microbiol. 1975; 29: 807
        • Thomas T.D.
        • Mills O.E.
        Proteolytic enzymes of starter bacteria.
        Netherlands Milk Dairy J. 1981; 35: 255
        • Trepman E.
        • Chen R.F.
        Fluorescence stopped-flow study of the ophthaldialdehyde reaction.
        Arch. Biochem. Biophys. 1980; 204: 524
        • Vakaleris D.G.
        • Price W.V.
        A rapid spectrophotometric method for measuring cheese ripening.
        J. Dairy Sci. 1959; 42: 264
        • Visser S.
        Proteolytic enzymes and their action on milk proteins. A review.
        Netherlands Milk Dairy J. 1981; 35: 65