Automated isolation of high-purity plasma albumin for isotope ratio measurements.
Measurement of the incorporation of labeled amino acids in plasma albumin, isolated from plasma sampled at different time points after infusion start is a well-known technique to study human albumin synthesis. Unfortunately, no chromatographic method has been described yet, enabling the automated isolation of high-purity albumin from large numbers of plasma samples as is required to study the kinetics of this process. Therefore, we developed a fast protein liquid chromatographic method, capable of processing 200 microliters amounts of plasma in 74 min (injection to injection). The system can run unattended as the FPLC system is connected to a sample processor equipped with a polyether ether ketone (PEEK) sample loop and a cooled sample tray. Albumin isolation was divided into three steps. First, plasma samples were injected onto a 1-ml Blue Sepharose HiTrap affinity column, equilibrated with 50 mmol/l phosphate buffer (pH 7.0). After elution of non-binding protein, switching the solvent to phosphate buffer with 1.5 mol/l sodium chloride eluted albumin. The resulting albumin fraction was desalted on-line by directing it through two consecutive HiTrap 5-ml desalting columns, whereafter it was retained in the system within a 5-ml PTFE loop, connected to a motor value. After switching this valve, thus bypassing the sample loop, the phosphate buffers were changed automatically to Tris buffers. Final purification involved elution of the captured fraction over a 1-ml ion-exchange Resource Q column, using a sodium chloride gradient, ranging from 0 to 0.5 mol/l in Tris buffer (20 mmol/l, pH 7.5). A more than 99% purity of the final albumin fraction was confirmed by capillary electrophoresis.
van Eijk
H M
HM
Department of Surgery, Maastricht University, The Netherlands.
Rooyakkers
D R
DR
van Acker
B A
BA
Soeters
P B
PB
Deutz
N E
NE
D001331:Automation; D002846:Chromatography, Affinity / Q000379:methods; D019075:Electrophoresis, Capillary; D006801:Humans; D007554:Isotopes; D012709:Serum Albumin / Q000737:chemistry / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet
D007554:Isotopes; D012709:Serum Albumin