Calibration of albumin-fatty acid binding constants measured by heptane-water partition.
Most measurements of binding affinity of albumin for long-chain fatty acids are based on heptane-water partition. In this method, equilibrium partition of fatty acid between heptane and an albumin-containing buffer is calibrated using the partition ratio between heptane and buffer in the absence of protein. In the current study, we used a variety of techniques to examine potential problems with this approach. Hydrophobic impurities in commercial [3H]palmitate preparations were incompletely removed by standard purification techniques. These impurities contributed from 5% of the total radioactivity in the heptane phase at low albumin concentrations (5 microM) to 62% at higher albumin concentrations (500 microM), thus confounding determination of binding affinity. These were identified by gas chromatography/mass spectroscopy as radio-labeled glycerol monopalmitate and monostearate. When albumin was not present, the partition ratio was similar to values reported by others. However, our results varied by a factor of four (265-1,119) depending on how the solutions were prepared. Although a true equilibrium partition must not depend on starting conditions, the partition ratio after 24-72 h was > 2x as large when tracer [3H]palmitate was added to the heptane phase than when it was added to the aqueous phase. Results also depended on the relative volumes of heptane and buffer used, approaching a maximum of 1,445 +/- 112 for very low heptane/buffer volume ratios. Much of this variability was due to hydrophilic impurities in [3H]palmitate, which ranged from 0.2 to 1.2% in commercial lots down to 0.1-0.5% after alkaline ethanol extraction and < 0.05% after thin-layer chromatography (TLC).(ABSTRACT TRUNCATED AT 250 WORDS)
Burczynski
F J
FJ
Department of Medicine, University of California, San Francisco 94143-0538.
Pond
S M
SM
Davis
C K
CK
Johnson
L P
LP
Weisiger
R A
RA
D002138:Calibration; D002855:Chromatography, Thin Layer; D005227:Fatty Acids / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006536:Heptanes; D008722:Methods; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000302:isolation & purification; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000378:metabolism*; D013997:Time Factors; D014867:Water
D005227:Fatty Acids; D006536:Heptanes; D010169:Palmitic Acids; D012709:Serum Albumin; D014867:Water; D019308:Palmitic Acid