Browsing by Author "Liau, CY"

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beta-Lactoglobulin is a thermal marker in processed milk as studied by electrophoresis and circular dichroic spectra
(2005-05-01)
Chen, WL
;
Hwang, MT
;
Liau, CY
;
Ho, JC
;
Hong, KC
As much of the sterilization process involves heat treatment during the preparation of milk on an industrial scale, the unpredictable measures of the process are an essential issue in determining the quality of the milk. The purpose of the present study was to investigate the major protein change(s) of whey proteins in processed milk and extend the knowledge for future reference in the dairy industry. Using a native polyacrylamide gel electrophoresis, we showed almost a 90% loss and denaturation of beta-lactoglobulin (LG), but not alpha-lactalbumin ( LA), in some brands of the processed and dry milks. Immunochemical analysis using Western blotting revealed that part of the loss was attributed to the formation of large multiple forms of LG in the processed product. Such denaturation was presumably associated with the heating procedure used in the process. Essentially, LG was the only major fraction converted to aggregates in milk heated at 95 degrees C for 30 min on 2-dimensional PAGE. The detailed thermal denaturation of purified LG and LA at various temperatures ( 50 to 95 degrees C) and time ( 5 to 960 s) were investigated using a circular dichroic analysis. The maximal changes of ellipticity at 205 nm ( converting beta-structure to disordered structure) were correlated to heating temperature and time. There were no significant conformational changes of LG at temperatures below 70 degrees C for as long as 480 s. Pronounced and rapid changes occurred between 80 to 95 degrees C in a time-dependent manner. Fifty percent of the maximal changes could be reached within 15 s. In conclusion, the unique chemical and immunochemical loss and conformational changes made LG a superior marker for evaluating the thermal processing of milk. The detailed thermal denaturation curves of LG constructed with its time and temperature in this study provide a valuable reference for the dairy industry. We postulate that heat treatment over 80 degrees C in 15 s may induce a significant denaturation of milk LG.
Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography
(2003-06-25)
Liau, CY
;
Chang, TM
;
Pan, JP
;
Chen, WL
;
Mao, SJT
Haptoglobin (Hp) is an acute-phase protein; its plasma levels increase consistently in response to infection and inflammation. The concentration of human plasma Up is ranged between 1 and 1.5 mg/ml. Similar to blood type, individual human Hp is classified as Hp 1-1, 2-1, or 2-2. The structural and functional analysis of the Hp, however, has not been studied in detail due to its difficult isolation procedure. Previously, we reported a single step for the purification of porcine Hp. In this study, we established a purification method using a high capacity hemoglobin-affinity column. Briefly, DEAE-purified human hemoglobin was first coupled to Sepharose 4B to prepare an affinity column in a 15-ml bed volume. Following a flow through of human plasma and an extensive wash, the bound material was eluted with a solution of 0.15 M NaCl, pH 11 (adjusted by ammonium), to remove low-affinity bound proteins. The high-affinity bound Hp was then eluted with 0.15 M NaCl containing 5 M urea, pH 11, and collected in tubes containing 100 mul of 1 M Tris buffer, pH 7.0. The biological activity of dialyzed Hp was retained as it formed a complex with hemoglobin on, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure, approximately 10 mg of Hp 1-1, with homogeneity greater than 96%, was obtained from 15 nil of human plasma. Affinity purified Up 2-1 or 2-2, however, contained trace amounts of apoA-I with the similar approach. The Hp could be further purified by HPLC using a Superose 12 gel-permeation chromatography, if desired, to achieve 100% purity. All the phenotypes of purified Hp consisted of alpha and beta chains on SDS-PAGE in the presence of a reducing reagent, further confirmed by a Western blot analysis. We conclude that human hemoglobin-affinity column was most suitable for the isolation of Hp 1-1 in large quantities. Whereas, one additional step using a gel-permeation was necessary for that of Hp 2-1 and 2-2. (C) 2003 Elsevier Science B.V. All rights reserved.