Sera and urine from patients with severe uremia and healthy persons were separated by means of gel permeation chromatography on Sephadex G-15 column with N（C2H5）3-H2CO3 buffer as the eluent. Two middle molecular peaks（A and B） were detected at 206 nm in normal urine, uremic serum and uremic urine, but these two peaks were hardly observed in the profile of normal sera. In contrast, the absorption at 206 nm of fractions A and B from uremic urine were smaller than that of fractions A and B from normal urine. Fractions A from normal urine, uremic serum and urine were collected and resolved into 3 subpeaks at 254 nm by high performance liquid chromatography. Two of these subpeaks, A-Ⅰ and A-Ⅱ, were detected in uremic serum, normal urine and uremic urine. The results of MALDI-TOF-MS revealed that the fraction A-Ⅰ from both uremic serum and normal urine contained a component with molecular weight {1 214}, which could hardly be seen in the fraction A-Ⅰ of uremic urine.
A uremic toxic compound with molecular weight 1007.94 was determined to be an octapeptide by mass spectrometry.Its amino-acid sequence was given as follos: Val-Val-Arg-Gly-Cys-Thr-Trp-Trp.Spin systems for amino acid residues in the octapeptide were identified through analysis of 2D NMR 1H-1H DQF-COSY,TOCSY and ROESY spectra acquired in H2O and D2O.Moreover,the complete assignment of proton resonances for the backbone and side chain was achieved.Based on the secondary chemical shift（Δδ） of the residues,the secondary structure of octapeptide was surveyed.Conformational analyses according to Chemical Shift Index（CSI） showed that the secondary structure of the octapeptide was principally α-helix.The CD spectra of the peptide in aqueous solution gave the same result.Additions of linear polymers made the conformations of octapeptide stretch.These experimental results provide a basis for further comprehension of interaction regulation between biomacromolecule and polymer absorbing materials.
Sera from patients with different courses of chronic renal failure and healthy persons were separated by gel permeation chromatography,and the concentration of middle molecular fraction B were calculated.The concentration of fractions B was increasing with the aggravating of renal failure degree,which indicated that the concentration of fraction B was related to patients′ renal failure.Fractions B from uremic urine,uremic sera or normal urine were resolved into 10 to 17 sub-fractions by ion exchange chromatography.Sub-fractions from normal urine and from uremic sera were assayed for their inhibition for the activity of Na,K-ATPase.The results showed that the Na,K-ATPase activities of the samples with B-8 and B-9 were much lower than that of blank.It is indicated that B-8 and B-9 contained some compounds which could inhibit the activity of Na,K-ATPase.
