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J. Biol. Chem., Vol. 280, Issue 5, 3574-3582, February 4, 2005

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Epitope Mapping of a Monoclonal Antibody Specific to Bovine Dry Milk

INVOLVEMENT OF RESIDUES 66–76 OF STRAND D IN THERMAL DENATURED -LACTOGLOBULIN^*

Chun Ying Song, Wen Liang Chen, Ming Chi Yang, Jen Pin Huang, and Simon J. T. Mao¶

From the Research Institute of Biochemical Engineering, Department of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu and Genesis Biotech Inc., 131, Lane 235, Bao-Chiao Road, Hsintien, Taipei, Taiwan, Republic of China

-Lactoglobulin (-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured -LG. In the present study, we show a dramatic increase in -LG immunoreactivity when heating raw milk between 70 and 80 °C. To map out the specific epitope of -LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66–76) of -LG. Circular dichroic spectral analysis shows that carboxymethylation on -LG not only resulted in a substantial loss of -configuration but also exerted a 10 times increase in immunoreactivity as compared with heated -LG. The result suggests that a further disordered structure occurred in -LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated -LG and its binding to retinol or palmitic acid. Most interestingly, pH 9–10, which neutralizes the Lys groups of -LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of -LG participated in the thermal denaturation between 70 and 80 °C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.

Received for publication, June 23, 2004 , and in revised form, November 1, 2004.

^* This work was supported by Grants NSC 90-2313-B-009-001, NSC 91-2313-B-009-001, NSC 92-2313-B-009-002, and NSC 93-2313-B-009-002 from the National Science Council of Taiwan, Republic of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai St., Hsinchu, Taiwan, Republic of China. Tel.: 886-3-571-2121 (ext. 56948); Fax: 886-3-572-9288; E-mail: mao1010{at}ms7.hinet.net.

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