A new method for purification of human plasma retinol-binding protein and transthyretin.
Retinol is transported in the blood bound to a specific carrier protein called retinol-binding protein (RBP), which in turn binds to another protein, transthyretin (TTR), a homotetrameric, thyroid-hormone-transporting protein. Binding of TTR increases the apparent molecular mass of RBP and thereby prevents glomerular filtration of RBP. Owing to their rapid turnover rates, plasma concentrations of these proteins are sensitive indicators and valuable diagnostic markers of vitamin A nutrition, protein energy malnutrition, infection and renal-tubule function. Previously RBP and TTR were purified by using different procedures, either from plasma or from pathological urine of humans. In general the procedure for purification of RBP and TTR is laborious, and extensive sample recycling is necessary for purification in appreciable amounts. In the present study, we have purified RBP using a simple method, which involves (NH(4))(2)SO(4) fractionation followed by sequential gel filtration under native conditions and 6 M urea. TTR, which was eluted in 60 kDa fractions during urea/Sephadex G-100 chromatography, was further purified to homogeneity using a combination of two dye-affinity chromatographic steps on Reactive Yellow and Cibacron Blue coupled to agarose columns. SDS/PAGE and immunoblotting, apart from typical UV absorption and fluorescent properties of RBP, were used for characterizing the purified RBP and TTR. Furthermore, the purified RBP and TTR were found to be functional from mutual binding monitored by fluorescence quenching.
Raghu
Pullakhandam
P
Department of Biophysics, National Institute of Nutrition, Indian Council of Medical Research, Jamai-osmania, Hyderabad-500 007, India.
Ravinder
Punjal
P
Sivakumar
Bhattiprolu
B
D000328:Adult; D002352:Carrier Proteins / Q000097:blood; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008297:Male; D011228:Prealbumin / Q000737:chemistry / Q000302:isolation & purification*; D012177:Retinol-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D054839:Retinol-Binding Proteins, Plasma
D002352:Carrier Proteins; D011228:Prealbumin; D012177:Retinol-Binding Proteins; D054839:Retinol-Binding Proteins, Plasma