The purpose of this study was to assess the possibility of isolating biologically active peptides from human blood using large volumes of blood filtrate, which are available from patients undergoing extracorporeal ultrafiltration because of renal insufficiency. This filtrate was submitted to six chromatographic separation steps, yielding one purified peptide which was completely analysed in its primary structure. It was found to be strikingly similar to proteins, described initially as rabbit uteroglobin (or blastokinin) and, more recently, from human bronchial lavage as the '10 kDa Clare cell protein', as well as from human urine as 'protein-1'. The natural molecule contains two chains of identical amino acid sequences of 70 residues which are arranged as an antiparallel dimer due to the disulphide bonds between two cysteines at positions 3 and 69. Mass analysis of the molecular forms yielded molecular weights from 15827 Da (non-oxidized form) to 15859 Da (bi-oxidized form). We conclude that this peptide isolated from the filtrate represents the human uteroglobin, and we demonstrate for the first time that this peptide may be involved as a humoral factor in reproductive or other physiological functions.

The purpose of this study was to assess the possibility of isolating biologically active peptides from human blood using large volumes of blood filtrate, which are available from patients undergoing extracorporeal ultrafiltration because of renal insufficiency. This filtrate was submitted to six chromatographic separation steps, yielding one purified peptide which was completely analysed in its primary structure. It was found to be strikingly similar to proteins, described initially as rabbit uteroglobin (or blastokinin) and, more recently, from human bronchial lavage as the ‘10 kDa Clara cell protein’, as well as from human urine as ‘protein-1’. The natural molecule contains two chains of identical amino acid sequences of 70 residues which are arranged as an antiparallel dimer due to the disulphide bonds between two cysteines at positions 3 and 69. Mass analysis of the molecular forms yielded molecular weights from 15 827 Da (non-oxidized form) to 15 859 Da (bi-oxidized form). We conclude that this peptide isolated from the filtrate represents the human uteroglobin, and we demonstrate for the first time that this peptide may be involved as a humoral factor in reproductive or other physiological functions.

4To whom correspondence should be addressed The purpose of this study was to assess the possibility of isolating biologically active peptides from human blood using large volumes of blood filtrate, which are available from patients undergoing extracorporeal urtrafirtration because of renal insufficiency. This filtrate was submitted to six chromatographic separation steps, yielding one purified peptide which was completely analysed in its primary structure. It was found to be strikingly similar to proteins, described initially as rabbit uteroglobin (or blastokinin) and, more recently, from human bronchial lavage as the '10 kDa Clara cell protein', as well as from human urine as 'protein-1'. The natural molecule contains two chains of identical amino acid sequences of 70 residues which are arranged as an antiparallel dimer due to the disulphide bonds between two cysteines at positions 3 and 69. Mass analysis of the molecular forms yielded molecular weights from 15 827 Da (non-oxidized form) to 15 859 Da (bi-oxidized form). We conclude that this peptide isolated from the filtrate represents the human uteroglobin, and we demonstrate for the first time that this peptide may be involved as a humoral factor in reproductive or other physiological functions.

A new method for the selective and quantitative determination of phosphotyrosine residues is presented using a differential iodination technique. Characterization of tyrosine-phosphorylated proteins was performed in a biological system using human U937 myeloid leukemia cells. The method is based on the saturation of free iodine binding sites using non-radioactive iodine. Samples are then treated with alkaline phosphatase. New iodine binding sites in dephosphorylated tyrosines are subsequently radio-iodinated, resulting in specific labeling of tyrosine phosphates. Separation is performed by RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled proteins are then identified using a radioactivity detector or autoradiography.

Specific labeling of tyrosine sulfate-containing peptides was achieved using a differential iodination approach. In a complex peptide mixture from human hemofiltrate, cold iodination to saturate free iodine binding sites was followed by mild acidic desulfation of tyrosine sulfate and subsequent radioiodination using iodine-125. Reaction steps were controlled by amino acid analysis using o-phthaldialdehyde precolumn derivatization and by spiking with a sulfated cholecystokinin fragment (CCK4-S). Separation of the peptide mixture with RP-HPLC on a C18 column coupled to a radioactivity monitor led to the sensitive (< or = 5 pM) and specific determination of tyrosine sulfate-containing peptides.

Several degradation products of fibrinogen have been shown to possess regulatory functions. Using peptide exacts from human blood filtrate, a large number of fibrinogen Aα fragments was identified. These fragments are generated at known plasmin attack sites and at several novel cleavage sites especially at hydrophobic and basic amino acid residues. One fragment containing the cell attachment site (RGD sequence) of fibrinogen Aα efficiently inhibits fibrinogen binding and platelet aggregation (IC50: 20-50 μM) in vitro. We conclude that in vivo degradation of fibrinogen Aα results in generation of endogenous antithrombotic peptides with local importance in fibrinolysis and platelet aggregation.

A sequential approach was developed to label tyrosine sulfate and peptides containing tyrosine sulfate selectively. Amino acids and peptides containing tyrosine and tyrosine sulfate were first iodinated using chloramine-T-method. Reaction products were determined by RP-HPLC. Mono- and biiodination of tyrosine and several model peptides was achieved within 120 s incubation time. Iodination of free tyrosine sulfate and sulfated cholecystokinin26-33 was less than 5%. After desulfation of the reaction products with 1 N HCl successful radioiodination of desulfated tyrosine was carried out whereas tyrosine did incorporate radioactive iodine only 10%. As shown by RP-HPLC specific labeling of tyrosine sulfate containing peptides with 125iodine was achieved.

A sequential approach was developed to label tyrosine sulfate and peptides containing tyrosine sulfate selectively. Amino acids and peptides containing tyrosine and tyrosine sulfate were first iodinated using chloramine-T-method. Reaction products were determined by RP-HPLC. Mono- and biiodination of tyrosine and several model peptides was achieved within 120 s incubation time. lodination of free tyrosine sulfate and sulfated cholecystokinin26-33 was less than 5%. After desulfation of the reaction products with 1 N HCl successful radioiodination of desulfated tyrosine was carried out whereas tyrosine did incorporate radioactive iodine only 10%. As shown by RP-HPLC specific labeling of tyrosine sulfate containing peptides with iodine-125 was achieved.

Human hemofiltrate (HF) was evaluated regarding its content of free amino acids, proteins, and regulatory peptides. Human HF was obtained from patients with end stage renal disease (ESRD). In contrast to plasma it mainly contains low and middle weight molecules < or = 45 kDa. The content of free amino acids, peptides, and proteins in pooled filtrate was determined by amino acid analysis using ortho-phthaldialdehyde/fluorenyl methyl chloroformate (OPA/FMOC) precolumn derivatization. The total amount of peptides and proteins in human HF is 49.4 mg/L (n = 8). The levels of all free amino acids (230 mg/L) and the concentration of some regulatory peptides like insulin, endothelin, gastrin, vasopressin and angiotensin II were similar compared with blood plasma. The amount of peptides and proteins detected in the filtrate was around 0.07% of total plasma proteins, and consisted mainly of smaller proteins and peptides as shown by size exclusion chromatography (SEC). The presence of large proteins in plasma is reduced by a factor of 1500 after filtration. We conclude that human hemofiltrate is a valuable source for the large-scale extraction of regulatory peptides.