Abstract
Abstract Prealbumin was isolated from human plasma by ammonium sulfate fractionation, chromatographic separations on diethylaminoethyl-Sephadex, and gel filtrations. The highly purified protein was subjected to quantitative amino acid analyses, which showed a minimum of 140 amino acid residues per prealbumin molecule. By determination of the minimum in the curves for the fraction of maximum deviation it was found that the minimum molecular weight for prealbumin was 15,500. Molecular weight determinations by sedimentation equilibrium ultracentrifugations gave a value of 62,500 ± 2,200. These results indicate that prealbumin is composed of four subunits. Molecular weight determinations by gel chromatography in 6 m guanidine hydrochloride gave values of about 16,000 for prealbumin. By this method it was shown that the subunits are held together by noncovalent bonds. A trypsin digest of prealbumin was examined by peptide-mapping techniques and the number of peptides (15 to 19) detected was in good agreement with the total number of lysine and arginine residues calculated from the amino acid composition of a prealbumin tetramer consisting of four identical chains. The NH2-terminal sequence of the protein was shown to be uniquely Gly-Pro. Prealbumin, reduced and 14C-carboxymethylated, was subjected to tryptic digestion, and the radioactivity was used to trace and isolate the cysteine-containing peptides. Only one peptide contained radioactivity, and analysis of this peptide revealed the unique sequence Gly-Pro-Ser-Met-Val-Cys(Cm)-Lys. These data strengthen the view that preal-bumin is composed of identical subunits. The mode of dissociation of the prealbumin tetramer was investigated by sedimentation equilibrium ultracentrifugation in various concentrations of guanidine hydrochloride. Determinations of local weight and number average molecular weights were consistent with three species being involved in chemical equilibrium, i.e. monomers, dimers, and tetramers of the prealbumin subunits.
Highlights
MethodsA Number of peptides expected was based on the amino acid composition given, assuming a tetrameric structure with identical subunits for prealbumin
In view of the reported single binding site for thyroxine and the 1: 1 stoichiometry of the prealbumin-RBP complex, an investigation was undertaken to explore the nature of the prealbumin subunit structure in greater detail, as our previous findings had shown that prealbumin consisted of four polypeptide chains of similar size
This communication describes the isolation of prealbumin from human plasma
Summary
A Number of peptides expected was based on the amino acid composition given, assuming a tetrameric structure with identical subunits for prealbumin. Determinufions of Free &djhydryl Groups in Prealbumin-Free sulfhydryl groups were reacted with 5,5’-dithiobis(2-nitrobenzoic acid) both with and without denaturing agents as described under “Experimental Procedure.”. Only be demonstrated with this technique after exposure of the protein to denaturing solutions containing isopropanol
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