Isolation and study of a fragment of human serum albumin containing one of the antigenic sites of the whole molecule

1. A fragment of human serum albumin called `inhibitor' has been degraded by trypsin, and one of the degradation products, designated fragment F1, has been isolated. Fragment F1 has a molecular weight of 6600. It contains neither tyrosine nor tryptophan. It is not precipitated with rabbit anti-sera to human serum albumin. 2. Fragment F1 was coupled to p-aminobenzylcellulose to form an insoluble conjugate. Rabbit anti-(human serum albumin) antibodies reacting with fragment F1 were specifically adsorbed on this conjugate and were desorbed by glycine–hydrochloric acid buffer. The isolated antibodies are composed of γ-globulin and β₂-macroglobulin. 3. Human serum albumin and fragment F1 formed with 7s anti-(fragment F1) antibodies soluble complexes that were studied by passive haemagglutination, ultracentrifugation and electrophoresis. Fragment F1 was shown to contain only one of the antigenic sites of albumin molecule. The 7s anti-(fragment F1) antibodies were shown to be bivalent and monospecific.     

Isolation procedure and some properties of myeloperoxidase from human leucocytes.

1. A rapid isolation procedure with a high yield for pure myeloperoxidase (donor:H2O2 oxidoreductase, EC 1.11.1.7) from normal human leucocytes is described. The enzyme was solubilized from leucocytes with the detergent, cetyltrimethylammonium bromide, and purified to apparent homogeneity. The yield of the enzyme was 17% with an absorbance ratio A430nm/A280nm = 0.85. 2. The purified enzyme showed three isoenzyme bands after polyacrylamide gel electrophoresis; ultracentrifuge studies indicated one homogeneous band with a molecular weight of 144 000. After reduction of myeloperoxidase, sodium dodecyl sulfate gel electrophoresis resolved an intense band (63 000 daltons) and a weak band (81 000 daltons). 3. The carbohydrate content of the enzyme was at least 2.5%. Mannose, glucose and N-acetylglucosamine were present. The amino acid composition is reported. 4. The EPR spectrum exhibited a high-spin heme signal with rhombic symmetry (gx = 6.92, gy = 5.07 and gz = 1.95). Upon acidification this signal was converted into a signal with more axial symmetry (g perpendicular = 5.89). At high pH (9.5) the EPR spectrum of the enzyme only shows low-spin ferric heme resonances. The circular dichroism spectra of ferric and ferrous myeloperoxidase in the visible and ultraviolet region show maxima and minima in ellipticity.     

Isolation and some properties of a deoxyribonuclease from Turbo cornutus.

1. Four DNases were found in the dried liver extract of a top shell, Turbo cornutus. The major one was purified 120-fold by phosphocellulose column chromatography, sulfoethylcellulose column chromatography and gel-filtration on Sephadex G-150. The yield was 2.7%. 2. The enzyme activity was not affected by Mg2+ (10(-3)--10(-2)M), EDTA (10(-3)--10(-2)M), or NaCl (10(-1)M). It showed a pH optimum of 4.7--4.8. Ionic strength was found to be critical for the maximal activity. The isoelectric point was 8.5--9.0. On heating at 50 degrees C C for 5 min the enzymic activity fell to half the initial value. 3. The enzyme preparation degraded native as well as heat-denatured DNA, but not RNA. It degraded heat-denatured DNA endonucleolytically to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphate and 5'-hydroxy termini of oligonucleotides were investigated. At both the 3'- and 5'-terminal positions, purine nucleotides were predominant.     

Orientation of the water molecules of hydration of human serum albumin

Through contact-angle measurements with a number of liquids, on layers of hydrated human serum albumin (HSA), built on anisotropic ultrafilter membranes, the apolar, Lifshitz-van der Waals surface tension component, as well as the polar, electron-acceptor and electron-donor parameters of the hydrated layers could be determined. From these data, it was found that the degree of orientation of the water molecules of hydration of HSA is 98% in the first layer of hydration and 30% of the second layer. The water molecules of hydration are oriented with the H atoms closest to, and the O atoms farthest from, the protein surface.     

Bilirubin binding properties of pigeon serum albumin and its comparison with human serum albumin

Binding of bilirubin (BR) to pigeon serum albumin (PgSA) was studied by absorption, fluorescence and CD spectroscopy and results were compared with those obtained with human serum albumin (HSA). PgSA was found to be structurally similar to HSA as judged by near- and far-UV CD spectra. However, PgSA lacks tryptophan. Binding of BR to PgSA showed relatively weaker interaction compared to HSA in terms of binding affinity, induced red shift in the absorption spectrum of BR and CD spectral characteristics of BR-albumin complexes. Photoirradiation results of BR-albumin complexes also showed PgSA-bound BR more labile compared to HSA-bound BR.     

Isolation and characterization of methionine synthetase from human placenta

The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.     

Differential effects of cysteine and methionine residues in the antioxidant activity of human serum albumin

Antioxidant properties of human serum albumin (HSA) may explain part of its beneficial role in various diseases related to free radical attack. In the present study, the antioxidant role of Cys and Met was studied by copper-mediated oxidation of human low density lipoproteins and by free radical-induced blood hemolysis which essentially assessed metal-chelating and free radical scavenging activities, respectively. Mild conditions were set up to specifically modify Cys and Met residues by N-ethylmaleimide (NEM) and chloramine T treatments, respectively. We found that Met and Cys accounted for 40-80% of total antioxidant activity of HSA. Copper binding to HSA was decreased by about 50% with chloramine T treatment of Met whereas no change was observed after NEM treatment of Cys. Although other amino acid residues are likely to be involved in anti-/prooxidant properties of HSA, from our data, we propose that Cys chiefly works as a free radical scavenger whereas Met mainly acts as a metal chelator.     

Synthesis and biophysical properties of polymerized human serum albumin

The use of many plasma expanders (PEs) is often limited by undesirable side effects, such as red blood cell (RBC) aggregation (hydroxyethyl starch), nephrotoxicity (dextran), and extravasation (albumin). Despite its natural prevalence in the bloodstream, human serum albumin (HSA) can increase the risk of mortality when administered to patients with increased vascular permeability (i.e., patients suffering from burns, septic shock, and endothelial dysfunction). The harmful extravasation of HSA can be limited by polymerizing HSA to increase its molecular size. In this study, HSA was nonspecifically cross-linked with glutaraldehyde at different cross-link densities by varying the molar ratio of glutaraldehyde to HSA. The results of this study show that the weight-averaged molecular weight (MW), viscosity, and extent of RBC aggregation of polymerized HSA increases with increasing cross-link density, whereas the colloid osmotic pressure (COP) decreases with increasing cross-link density. Interestingly, circular dichroism measurements indicate that the secondary structure of HSA is unaffected by polymerization. Altogether, these results show that glutaraldehyde can effectively cross-link HSA to produce high MW polymers, yielding a novel series of potential PEs that exhibit low COP and high viscosity.     

Secretion of human serum albumin from Bacillus subtilis.

We have fused the structural gene (hsa) for human serum albumin (HSA) to the expression elements and signal sequence coding region of each of two genes from Bacillus amyloliquefaciens P, an alpha-amylase gene (amyBamP) and a neutral protease gene (nprBamP). Bacillus subtilis strains harboring either of these gene fusions synthesized a protein with the antigenic characteristics and size (68 kilodaltons) of HSA. Results from pulse-labeling studies indicated that the bacterially produced HSA was secreted from cells which had been converted to protoplasts. Results from similar studies with intact cells suggested that the signal sequence was removed from the hybrid protein, providing further evidence that B. subtilis can translocate this foreign protein across the cell membrane. Signal sequence removal was efficient when the level of HSA synthesis was low. However, in strains which synthesized HSA at a high level, signal sequence removal was less efficient.