Fractionation and Purification of Goat Milk Proteins: - by Preparative High-Pressure Liquid Chromatography
Translated title
Fraktionering og Oprensning af Proteiner fra Gedemælk: - ved brug af Preparativ High-Pressure Liquid Chromatography
Author
Madsen, Ditte Arnanguag
Term
7. term
Education
Publication year
2014
Submitted on
2014-04-22
Pages
40
Abstract
Formålet var at isolere rene fraktioner af tre proteiner fra gedemælk—αs1-kasein, α-laktalbumin og β-laktoglobulin—til brug som interne standarder i væskekromatografi, en teknik der adskiller og måler molekyler. En intern standard er et kendt referenceprotein, som bruges til at forbedre nøjagtighed og sammenlignelighed i målingerne. Disse proteiner har molekylmasser på henholdsvis 18191.27, 14194.12 og 23615.33 Dalton (UniProt, 2014), og de er ikke tilsvarende proteiner fra komælk. Kaseiner er mælkproteiner med et isoelektrisk punkt omkring pH 4,6; fordi de mangler udtalte foldede (sekundære og tertiære) strukturer, er de relativt varmestabile. Valleproteiner er de proteiner, der bliver i opløsning efter udfældning. Fraktionering og oprensning blev udført med præparativ højtryksvæskekromatografi (HPLC) og efterfølgende frysetørring. Renheden af fraktionerne blev vurderet med time-of-flight væskekromatografi-massespektrometri. Alle tre proteiner blev forsøgt oprenset, men inden for den tilgængelige tidsramme lykkedes det kun at opnå β-laktoglobulin med ≥95 % renhed: 11 mg fra 38.12 ml skummet gedemælk, svarende til cirka 10 % af det maksimale udbytte. Der blev desuden udført Bradford-proteinassay på både mælken og det oprensede protein. Dette viste, at Kjeldahl-metoden og Bradford-assayet ikke giver samme proteinkoncentration for den samme prøve; Kjeldahl gav konsekvent højere værdier end Bradford.
The aim was to isolate pure fractions of three goat milk proteins—αs1-casein, α-lactalbumin, and β-lactoglobulin—for use as internal standards in liquid chromatography, a technique that separates and measures molecules. An internal standard is a known reference protein used to improve accuracy and comparability in measurements. These proteins have molecular masses of 18191.27, 14194.12, and 23615.33 Daltons, respectively (UniProt, 2014), and they are not the same as the corresponding proteins from cow’s milk. Caseins are milk proteins with an isoelectric point around pH 4.6; because they lack extensive folded (secondary and tertiary) structures, they are relatively heat stable. Whey proteins are those that remain dissolved after precipitation. Fractionation and purification were performed using preparative high-pressure liquid chromatography (HPLC) followed by freeze-drying. The purity of the fractions was assessed with time-of-flight liquid chromatography mass spectrometry. All three proteins were targeted, but within the available timeframe only β-lactoglobulin was obtained at ≥95% purity: 11 mg from 38.12 ml of skimmed goat’s milk, which is about 10% of the maximum yield. A Bradford protein assay was also carried out on both the milk and the purified protein. This showed that the Kjeldahl method and the Bradford assay do not give the same protein concentration for the same sample; Kjeldahl consistently gave higher values than Bradford.
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