Effects of Long-term Storage on Human Serum Albumin. Ii. Follow-up of Chromatographically and Ultracentrifugally Detectable Changes.

This laboratory previously reported changes in normal human serum albumin stored under various conditions for 5 years. This report demonstrated that coordinated use of chromatography and ultracentrifugation offered a sensitive method for detecting protein alterations (1). In the present studies, which utilized the same method after another 5-year interval, the progress of these changes has been followed, and the relative stability of albumin stored under certain conditions has been determined. Methods The samples were vials of commercial normal human serum albumin, which was described previously (1). They had been prepared by low temperature ethanol fractiona-tion of citrated whole plasma or ultraviolet irradiated dried plasma and had been heated at 600 C for 10 hours in the presence of stabilizers. Storage was at 50 or 320 C. The samples stored in the dry state had been sealed in vacuo. Gradient elution chromatography of portions containing 500 mg of albumin was done at 6° C on 2.2-X 35-cm columns of the same diethylaminoethyl (DEAE) cellulose used in the earlier study (1). The gradient was from entire chromatographic procedure was that used previously (1), with the following exception. Whereas re-generation of the adsorbent had involved removing it from the column, washing with water, NaOH, water, warm ethanol, water, and starting buffer, followed by repacking, the present regeneration method consisted simply of flushing the column with approximately 400 ml of a solution that was 0.1 M in Na3PO4 and 0.2% (vol/ vol) in Triton X-100 2 (2) and subsequently flushing with at least 1,400 ml of starting buffer. Appropriate chromatographic fractions were concentrated by negative pressure dialysis against 0.9%o NaCl at 20 C, dialyzed against phosphate-buffered saline, pH 7.2, ionic strength 0.154, and centrifuged at 200 C in a Spinco model E ultracentrifuge at a protein concentration of 1.05%. They were then subjected to paper electrophoresis at room temperature in barbital buffer at pH 8.6. These procedures have been presented in detail previously (1); additional methods and any deviations from these procedures will be described below. Elution patterns exhibited by the various samples have been classed into five general types, I through V (Figure 1), based on the typical chromatographic profiles shown in the earlier publication (1). Designations of plus or minus following the Roman numeral have been used to indicate the presence of more or less region 2 than that illustrated. In the present experiments, the small "foot" previously designated region 3 was …