Unequal incorporation of amino acids into plasma albumin by the isolated perfused rat liver (original) (raw)
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A Comparative Study of Commercial Human and Bovine Albumin Preparations
Annals of Clinical Biochemistry, 1978
A number of commercial human and bovine albumin preparations were compared using seven assay procedures, to assess their suitability as reference materials for albumin and total protein assays. The results indicate that before a particular commercial albumin preparation can be used for standardisation purposes, its suitability should be checked in several assay systems which measure a different functional aspect of the protein molecule. The measurement of extinction coefficient in the range 278-280 nm does not appear to be a valid measure of protein content if the albumin preparation is to be used for standardisation of immunochemical or dye-binding assays. Reaction depends on certain amino-acid sequences. phenolic groups as in tyrosine, other ill defined reactants (see Chou and Goldstein, 1960) Absorbance due to aromatic amino-acids, mainly tyrosine, tryptophan, and phenylalanine Specific immunological groupings. Allllfegation aided and stabilised with polyethylene Slycol-6000. Automated system using Tcchnicon AutoAnalyzer Specific immunological groupings and electrodiffusion properties Specific immunological groupings and diffusion properties •Stock albumin solution diluted with 154 mmol/l NaCI to Ilive a measured absorbance within the range 0•35 to 0'50 in a 1•0 cm cuvette. Spectrophotometer Unicam SP 1800.
Clinical Science, 1984
1. The infusion of cationic substances produces acute renal failure and proteinuria, and an experimental disease very similar to disseminated coagulopathy. The purpose of this work was to investigate further, in the rat, the plasma disappearance rate, the tissue distribution and the catabolism of albumins with modified isoelectric points. 2. Human serum albumin was cationized with hexanediamine and labelled with 125I or 131I. 3. During 10–180 min after their intravenous injection into the rat, these modified 125I-labelled albumins were cleared from the plasma at a rate which increased with their isoelectric point. 4. At 1 and 3 h after the injection of highly cationic proteins (isoelectric point higher than 9.5) the tissue protein bound 125I concentration was greatest (∼3.5% of the injected activity/g) in the spleen and liver. A significant amount of the basic proteins was found in the kidney and in the lung (0.75–1% /g). Their concentration was much lower in other tissues. 5. The w...
Investigation of an albumin-enriched fraction of human serum and its albuminome
PROTEOMICS – Clinical Applications, 2007
The removal of albumin and other high abundance proteins is a routine first step in the analysis of serum and plasma proteomes. However, as albumin can bind proteins and peptides, there is a universal concern as to how the serum proteome is changed by the removal of albumin. To address this concern, the current study was designed to identify proteins and peptides removed from the serum during albumin depletion; to determine which of these are bound to albumin (rather than copurified) and whether the bound proteins are intact proteins or peptide fragments. Sequential, independent analyses including both anti-albumin antibody (anti-HSA) affinity chromatography and SEC were used to isolate albumin-bound proteins. RP-HPLC and 1-D SDS-PAGE were then used to further separate the proteins prior to identification by MS/MS. Finally, whole protein molecular weight (MW) MS measurements coupled with protein coverage obtained by MS were combined to assess whether the bound proteins were intact or peptide fragments. Combining the results from multiple approaches, 35 proteins, of which 24 are intact, were found to be associated with albumin, and they include both known high and low abundance proteins.
Molecular Immunology
Antisera were raised in rabbits against fragment 11-193 (less Arg-144) or fragment 37%571 of bovine serum albumin (BSA). Several rabbits and bleedings at different time intervals from each rabbit were studied. Fragment 377-571, corresponding to the last third of BSA, was invariably more immunogenic than fragment 11-193. Antisera to fragment 377-571 showed, in quantitative precipitin analysis, appreciable cross-reactions with BSA and with fragment 11-193, which increased with time after immunization. Similar results were obtained with antisera to fragment 11-193. The reactivity of antisera to fragment 377-571 with BSA was removed almost completely by absorption with fragment 11-193. Conversely, absorption of the antisera with BSA removed their reaction with fragment 11-193 almost entirely. Joint absorption by BSA and fragment 11-193, each at equivalence, gave a reaction similar to that obtained by either fragment 11-193 alone or BSA alone in the region of antigen excess. After absorption of antisera to fragment 377-571 with BSA, addition of fragment 11-193 at equivalence (relative to the unabsorbed antiserum) gave no immune precipitate but inhibited 100~ the precipitin reaction of the absorbed antisera with fragment 377-571. However, antibodies in these antisera were removed quantitatively on an immunoabsorbent of fragment 377-571. Similar findings were obtained in absorption studies on antisera to fragment 11-193. A BSA-immunoabsorbent removed quantitatively all the antibodies in whole antisera to fragment 377-571. Similarly, an immunoabsorbent of fragment 11-193 removed all these antibodies. Antibodies recovered from these immunoabsorbents did not react best in precipitin reaction with BSA or fragment 11-193, but with fragment 377-571. In this respect, they behaved exactly like the original whole antiserum and explained the observed differences in the precipitating ability of the antisera with fragments 11-193, 377-571 and BSA. These studies confirmed that BSA has repeating identical antigenic sites. Furthermore, fragments 377-571, 11-193 or BSA were essentially the same immunochemically but antisera directed against any of these as an immunogen differed only in their abilities to form insoluble immune complexes, and not in their abilities to bind with the other two cross-reacting antigens.
Patterns of albumin and general protein synthesis in rat liver as revealed by gel electrophoresis
Biochimica et biophysica acta, 1972
Patterns of protein synthesis in rat liver microsomal fractions have been studied using polyacrylamide gel electrophoresis. In vivo patterns were obtained after injecting rats with 14C-labelled amino acids, and sacrificing them 20 min later. The livers were fractionated by standard techniques. Synthesis of albumin by the liver microsomes was found to be somewhat reduced relative to other proteins, i8-2o h after partial hepatectomy. In vitro patterns were obtained by incubating subcellular fractions with 14Clabelled amino acids, GTP and an ATP-generating system, and examining the sonic extract of the incorporation mixture. Extracts from regenerating livers again made less albumin relative to other proteins than did normal extracts. In vivo-like patterns were obtained using the 500 ×g, 3ooo ×g, 15 ooo ×g supernatants, and microsomes with microsomal supernatant. Integrity of the pattern was lost upon dilution of the microsomal supernatant or when polysomes were used with whole supernatant. The major peak in the patterns co-electrophoresed slightly behind rat serum albumin and was identified as albumin by chemical and immunological means.
Study of the antigenic structure of human serum albumin with monoclonal antibodies
Molecular Immunology, 1985
Analysis of the antigenic structure of human serum albumin was undertaken using monoclonal antibodies. Nineteen antibodies were prepared and their specificities were studied using fragments which encompass the whole sequence of the albumin molecule. These antibodies recognized 13 different epitopes which are different from the one previously identified with two other monoclonal antibodies [Doyen et al., Immun. Left. 3, 365-370 (1981)]. Among those 13 different epitopes, six were overlapping. Four epitopes were located on the N-terminal half of the albumin molecule. One of these required integrity of methionine 87 and the other three were overlapping and located around methionine 123. Eight epitopes were located on the C-terminal half of the albumin. Two of them were within the sequence, 330-422 and 299-496 respectively; the other six appeared to be topographic determinants which were altered or lost in the albumin fragments. A last epitope could not be located on any region of albumin. Four monoclonal antibodies directed against a given portion of the albumin molecule reacted slightly with another part of albumin, thus confirming the existence of an intramolecular cross-reactivity between the different domains of human albumin.
The reactivity of human serum albumin toward trans‐4‐hydroxy‐2‐nonenal
2012
Mass spectrometry was used to probe the preferred locations of trans-4-hydroxy-2-nonenal (HNE) addition to the cysteine, histidine, and lysine residues of human serum albumin (HSA). Considering only those modified peptides supported by high mass accuracy Orbitrap precursor ion measurements (high confidence hits), with HNE:HSA ratios of 1:1 and 10:1, 3 and 15 addition sites, respectively, were identified. Using less stringent criteria, a total of 34 modifications were identified at the higher concentration. To gain quantitative data, iTRAQ labeling studies were completed. Previous work had identified Cys 34 , the only free cysteine, as the most reactive residue in HSA, and we have found that Lys 199 , His 242/7 , and His 288 are the next most reactive residues. Although the kinetic data indicate that the lysines and histidines can react at relatively similar rates, the results show that lysine addition is much less favorable thermodynamically; under our reaction conditions, lysine addition generally does not go to completion. This suggests that under physiological conditions, HNE addition to lysine is only relevant in situations where unusually high HNE concentrations or access to irreversible secondary reactions are found.
Clinical Biochemistry, 2000
Objectives: This study investigates the sensitivity of various standard clinical techniques in the detection of albumin fragments. The significance of this work is in the detection of urinary proteins, such as albumin, which has recently been discovered to be excreted as mainly peptide fragments as a result of filtered albumin undergoing degradation during renal passage. All filtered proteins undergo a similar degradation process. Design and Methods: Albumin digested with trypsin was used as a model urine solution. The solution was assayed for albumin concentration by various methods including the biuret assay that is known to detect urinary albumin fragments. The digest solution was also analyzed by various clinically used chromagen assays, electrophoretic and chromatographic methods to determine whether they are able to detect the fragmented protein.