Albumin is purified by dissolving it in conductivity water and passage at 2-4o through two ion-exchange columns, each containing a 2:1 mixture of anionic and cationic resins (Amberlite IR-120, H-form, Amberlite IRA-400, OH -form). This treatment removes ions and lipid impurities. Care is taken to exclude CO2, and the solution is stored at -15o. [M.ller et al. Trans Faraday Soc 57 312 1961.] More complete lipid removal is achieved by lyophilising the de-ionised solution, covering the dried albumin (human serum) with a mixture of 5% glacial acetic acid (v/v) in iso-octane (previously dried with Na2SO4) and allowing it to stand at 0o (without agitation) for upwards of 6hours before decanting and discarding the extraction mixture, washing with iso-octane, re-extracting, and finally washing twice with iso-octane. The purified albumin is dried under vacuum for several hours, then dialyzed against water for 12-24hours at room temperature, lyophilised, and stored at -10oC [Goodman Science 125 1296 1957]. It has been recrystallised in high (35%) and in low (22%) EtOH solutions from Cohn's Fraction V. The high EtOH recrystallisation is as follows: To 1kg of Fraction V albumin paste at -5o is added 300mL of 0.4 M pH (pH 5.5) acetate buffer in 35% EtOH pre-cooled to -10o and 430 mL of 0.1 M NaOAc in 25% EtOH also at -10o. Best results are obtained by adding all of the buffer and about half of the NaOAc and stirring slowly for 1hour. The rest of the NaOAc is added when all the lumps have disintegrated. The mixture is set aside at -5o for several days to crystallise. 35% EtOH (1 L) is then added to dilute the crystalline suspension and lower the ionic strength prior to centrifugation at -5o (yield 80%). The crystals are further dissolved in 1.5 volumes of 15% EtOH/0.02M NaCl at -5o and clarified by filtration through washed, calcined diatomaceous earth. This solution may be recrystallised by re-adjusting to the conditions in the first crystallisation, or it may be recrystallised at 22% EtOH with the aid of a very small amount of decanol (enough to give a final concentration of 0.02%). Note that crystallisation from lower EtOH concentration gave better purification (i.e. by removing globulins and carbohydrates) and producing a more stable product. The low EtOH recrystallisation is as follows: To 1kg of Fraction V at -10o to -15o is added 500mL of 15% EtOH at -5o, stirred slowly until a uniform suspension is formed. To the 15% EtOH (500mL) is added sufficient 0.2M NaHCO3 solution (125-150mL) at 0o to bring the pH (1:10 dilution) to 5.3. Some temperature rise occurs, and care must be taken to keep the temperature < -5o. If the albumin is incompletely dissolved a small amount of H2O is added (100mL at a time at 0o, allowing 15minutes between additions). Undissolved albumin can be easily distinguished from small amounts of undissolved globulins, or as the last albumin dissolves, the appearance of the solution changes from milky white to hazy grey-green in colour. Keep the solution at -5o for 12hours and filter by suspending in 15g of washed fine calcined diatomaceous earth, and filtering using a Büchner funnel precoated with coarser diatomaceous earth. The filtrate may require two or more similar filtrations to give a clear solution. To crystallise the filtrate, add through a capillary pipette, and with careful stirring, 1/100volume of a solution containing10% decanol and 60% EtOH (at -10o), and seed with the needle-type albumin crystals. After 2-3days, crystallisation is complete. The crystals are centrifuged off. These are suspended with gentle mechanical stirring in one-third their weight of 0.005 M NaCl pre-cooled to 0o. With careful stirring, H2O (at 0o) is added slowly in an amount equal to 1.7 times the weight of the crystals. At this stage there is about 7% EtOH, and the temperature cannot be made lower than -2.5o to -1o. Clarify, and collect as above. [Cohn et al. J Am Chem Soc 69 1753 1947.] Human serum albumin has been purified similarly with 25% EtOH and 0.2% decanol. The isoelectric points of bovine and human serum albumins are 5.1 and 4.9, respectively.
Suitable for cell culture media supplement.