Heat Treatment of Bovine -Lactalbumin Results in Partially Folded, Disulfide Bond Shuffled States with Enhanced Surface Activity
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- 作者：Ramani Wijesinha-Bettoni ; Chunli Gao ; John A. Jenkins ; Alan R. Mackie ; Peter J. Wilde ; E. N. Clare Mills ; Lorna J. Smith
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：August 28, 2007
- 年：2007
- 卷：46
- 期：34
- 页码：9774 - 9784
- 全文大小：503K
- 年卷期：v.46,no.34(August 28, 2007)
- ISSN：1520-4995
文摘
Prolonged heating of holo bovine 
rs/alpha.gif" BORDER=0>-lactalbumin (BLA) at 80 
C in pH 7 phosphate buffer inthe absence of a thiol initiator improves the surface activity of the protein at the air:water interface, asdetermined by surface tension measurements. Samples after 30, 60, and 120 min of heating were analyzedon cooling to room temperature. Size-exclusion chromatography shows sample heterogeneity that increaseswith the length of heating. After 120 min of heating monomeric, dimeric, and oligomeric forms of BLAare present, with aggregates formed from disulfide bond linked hydrolyzed protein fragments. NMRcharacterization at pH 7 in the presence of Ca2+ of the monomer species isolated from the sample heatedfor 120 min showed that it consisted of a mixture of refolded native protein and partially folded proteinand that the partially folded protein species had spectral characteristics similar to those of the pH 2 moltenglobule state of the protein. Circular dichroism spectroscopy showed that the non-native species hadapproximately 40% of the rs/alpha.gif" BORDER=0>-helical content of the native state, but lacked persistent tertiary interactions.Proteomic analysis using thermolysin digestion of three predominant non-native monomeric forms isolatedby high-pressure liquid chromatography indicated the presence of disulfide shuffled isomers, containingthe non-native 61-73 disulfide bond. These partially folded, disulfide shuffled species are largelyresponsible for the pronounced improvement in surface activity of the protein on heating.
rs/alpha.gif" BORDER=0>-lactalbumin (BLA) at 80 C in pH 7 phosphate buffer inthe absence of a thiol initiator improves the surface activity of the protein at the air:water interface, asdetermined by surface tension measurements. Samples after 30, 60, and 120 min of heating were analyzedon cooling to room temperature. Size-exclusion chromatography shows sample heterogeneity that increaseswith the length of heating. After 120 min of heating monomeric, dimeric, and oligomeric forms of BLAare present, with aggregates formed from disulfide bond linked hydrolyzed protein fragments. NMRcharacterization at pH 7 in the presence of Ca2+ of the monomer species isolated from the sample heatedfor 120 min showed that it consisted of a mixture of refolded native protein and partially folded proteinand that the partially folded protein species had spectral characteristics similar to those of the pH 2 moltenglobule state of the protein. Circular dichroism spectroscopy showed that the non-native species hadapproximately 40% of the rs/alpha.gif" BORDER=0>-helical content of the native state, but lacked persistent tertiary interactions.Proteomic analysis using thermolysin digestion of three predominant non-native monomeric forms isolatedby high-pressure liquid chromatography indicated the presence of disulfide shuffled isomers, containingthe non-native 61-73 disulfide bond. These partially folded, disulfide shuffled species are largelyresponsible for the pronounced improvement in surface activity of the protein on heating.