Purification of a-lactalbumin

Flowchart for isolation and purification of a-lactalbumin from milk. 

What other purification techniques could be applied to the purification of a-lactalbumin Use a MEDLINE literature search. Study the following references for methods that have been applied to the isolation and purification of a-lactalbumin W. G. Gordon and W. F. Semmett,/. Atner. Cbem. Soc. 75,328 (1953). W. G. Gordon and J. Ziegler, Biochem. Prep. 4,16 (1955). U. Brodbeck, W. L. Denton, N. Tanahashi, and K. E. Ebner,/ Biol. Cbem. 242,1391 (1967). F. J. Castellino and R. L. Hill,/. Biol. Cbem. 245, 417 (1970). L. Lindahl and H. Vogel, Anal. Biocbem. 140,394(1984). 

Most proteins have a broad characteristic absorption spectrum centered at about 280 nm. The major absorption is due to the presence of aromatic moieties in the amino acids phenylalanine, tyrosine, and tryptophan. During the a-lactalbumin purification described in this experiment, you will monitor the process by measuring the absorption at 280 nm (A2S0) of column fractions to be sure the experiment is proceeding correctly. You must recognize that you are measuring not the concentration or presence of a-lactalbumin specifically but the total amount of all proteins present. 

The complete isolation, purification, and characterization of a-lactalbumin as described here require about 9 hours. The preparation of whey is completed in approximately 3 hours and the chromatographic step (Sephadex or affinity) requires another 3 hours. The analysis procedures require 3 hours. The whey fraction may be stored in a freezer for several weeks if desired. As an alternative to isolation, students may be provided commercial a-lactalbumin, which is then further purified by chromatography or analyzed directly by SDS-PAGE. The time for gel electrophoresis can be greatly reduced if commercially prepared gel plates are used. 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

Arunkumar, A. and M. R. Etzel. 2013. Fractionation of a-lactalbumin from P-lactoglobulin using positively charged tangential flow ultrafiltration membranes. Sep. Purif. Technol. 105 121-128. 

Barman, T. E. 1970. Purification and properties of bovine milk glyco-a-lactalbumin. Biochim. Biophys. Acta 214, 242-243. 

L Hall and P Campbell, in Essays in Biochemistry, Vol 22, R. Marshall and K Tip-ton, Editors (1988), Academic Press (London), pp 1-26. A review on a-lactalbumin E Harris and S. Angal, Editors, Protein Purification Applications A Practical Approach, Vol 2 (1990), IRL Press (Oxford) An excellent book for undergraduates B. Johnson, The Scientist, November 9, 1998 A review of cell homogenizers A Lehmnger, D. Nelson, and M Cox, Principles of Biochemistry, 3rd ed (1999), Worth Publishers (New York), pp 130-133 Working with proteins. 

Lindahl and Vogel (1984) studied the purification of bovine, human, caprine, ovine, and equine a-lactalbumins, exploiting the property of... 

An important development in the purification of galactosyltransferase has been the use of either a-lactalbumin or a substrate, bound to an immobile inert substance in an affinity column. With a-lactalbumin, either glucose or A-acetylglucosamine is included in the buffer to enhance the binding of galactosyltransferase to a-lactalbumin. The galactosyltransferase is then eluted with buffer not containing saccharide. 

Andrews (1970) and Trayer and Hill (1971) were apparently the first to use a-lactalbumin, bound to Sepharose in an affinity column, for the purification of galactosyltransferase. Trayer et al. (1974) used a-lactalbumin—agarose as a specific adsorbent for galactosyltransferase. 

Jeng, S.-Y., Bieck, G.T., Wheeler, M.W., and Jimenez-Flores, R. (1997). Characterization and partial purification of bovine a-lactalbumin and /1-casein produced in milk of transgenic mice. J. Dairy Sci. 80,3167-3175. 

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