Goat Serum Albumins Research Articles

Computational study to find the goat serum albumin (GSA) binding site as A-esterase

A-esterases are a classical term applied to enzymatic activity of the proteins by a mechanism not involving intermediate covalent phosphorylation, but requiring a divalent cation cofactor. Recently, a copper-dependent A-esterase activity has been identified in goat serum albumin (GSA) on the organophosphorus insecticide trichloronate. This hydrolysis was identified ex vivo with spectrophotometry and chromatography techniques. Albumin mechanism of action and catalytic site as Cu2+-dependent A-esterase are still unknown. Therefore, to know the copper bind to albumin is relevant. N-terminal sequence has been reported as the high affinity site for this cation, due to the histidine in position 3. The aim of this work in silico is to explore how occurs this metallic binding and active the esterase catalytic function. The GSA crystallized structure (PDB: 5ORI) was chosen for molecular docking and dynamics. A site-directed docking, for N-terminal site and a blind docking was done with trichloronate as ligand. Root-mean-square deviation and frequency plot was calculated to find the most frequent predicted structure and visualize the amino acids involved in binding site. The affinity energy in the blind docking (−5.80 kcal/mol) is almost twice lower than site-directed docking (−3.81 kcal/mol) and N-terminal amino acids do not appear in the most repeated structure binding site, suggesting that the protein has a site with higher affinity to the trichloronate ligand. His145 could be involved in the binding site as has been reported in previous studies.

Cu2+-dependent stereoselective hydrolysis of a chiral organophosphonothioate insecticide for domestic mammals’ sera and its albumins

Acute toxicity of organophosphate (OPs) pesticides is a public health problem. The adverse effects are associated with the inhibition and aging of nervous system B-esterases such as acetyl cholinesterase (AChE) and neuropathic target esterase (NTE). Treatment based on A-esterases such as mammal serum paraoxonase-1 has been suggested. This ex vivo study shows the Cu2+-dependent hydrolysis of trichloronate (TCN), a racemic organophosphonothioate insecticide, in human and domestic mammal serum (dog, goat, pig, sheep and cow). Ca2+-dependent (2.5 mM) or EDTA-resistant (5 mM) activity (1–6%) was not significant (p>0.05) in all samples, except goat serum and its albumin, which showed higher levels of TCN hydrolysis (38–58%) than other mammals with 100 and 300 μM copper sulfate at physiological conditions for 60 min. Goat serum albumin (GSA) showed significant (p˂0.05) stereoselective hydrolysis (+)-TCN ˃ (−)-TCN (45% versus 33%). This suggests that GSA is the protein responsible for Cu2+-dependent TCNase activity in goat serum. This is the first report on Cu2+-dependent A-esterase activity in mammalian tissues. This goat serum cuproprotein could be considered as an alternative in future biotechnological applications including enantiomeric synthesis, bioremediation and antidotal treatment of organophosphonothioate pesticide poisoning.

Exploring ligand-protein interaction: A laboratory exercise on herbicide binding to plasma transport protein.

A laboratory exercise on the interaction between the herbicide pendimethalin (PM) and goat serum albumin (GSA), a carrier protein present in mammalian blood circulation, is described. Fluorescence spectroscopy was used to study the binding reaction between PM and GSA. Titration of a constant amount of the protein (GSA) with increasing ligand (PM) concentrations produced a consecutive decrease in the protein's fluorescence. Treatment of the fluorescence quenching data according to the Stern-Volmer equation yielded the values of the Stern-Volmer constant (Ksv ) and bimolecular quenching rate constant (kq ), whereas values of the binding constant (Ka ) and number of binding sites (n) were obtained from the double logarithmic plot. This experiment provides an exciting opportunity for undergraduate students to independently perform ligand binding studies with a protein, in addition to providing the means for the determination of their binding parameters. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 156-160, 2019.

Chiral Recognition of Mandelic Acid with Bovine and Goat Serum Albumins by Flow Injection Analysis-Quartz Crystal Microbalance

Chiral Recognition of Mandelic Acid with Bovine and Goat Serum Albumins by Flow Injection Analysis-Quartz Crystal Microbalance

A New Biosensor for Chiral Recognition Using Goat and Rabbit Serum Albumin Self‐Assembled Quartz Crystal Microbalance

Quartz crystal microbalance (QCM) biosensor was used for the chiral recognition of five pairs of enantiomers by using goat serum albumin (GSA) and rabbit serum albumin (RbSA) as chiral selectors. Serum albumin (SA) was immobilized on the QCM through the self-assembled monolayer technique, and the surface concentration of GSA and RbSA were 8.8 × 10(-12) mol cm(-2) and 1.2 × 10(-11) mol cm(-2) , respectively. The QCM biosensors showed excellent sensitivity and selectivity. Meanwhile, the chiral recognition of SA sensors was quite species dependent. There were differences between GSA and RbSA sensors in the ability and the preference of chiral recognition. To R,S-1,2,3,4-tetrahydro-1-naphthylamine (R,S-1-TNA), R,S-1-(4-methoxyphenyl)ethylamine (R,S-4-MPEA), and R,S-1-(3-methoxyphenyl)ethylamine (R,S-3-MPEA), the preference of the stereoselective SA-drug binding of the two kinds of SA sensors were consistent. However, to R,S-2-octanol (R, S-2-OT) and R,S-methyl lactate (R,S-MEL), the two kinds of SA sensors had opposite chiral recognition preference. Moreover, the interactions of SA and the five pairs of enantiomers have been further investigated through ultraviolet (UV) and fluorescent (FL) spectra. The UV/FL results were in accordance with the consequence of QCM.

The Pro-inflammatory Mediator Leukotriene D4 Induces Phosphatidylinositol 3-Kinase and Rac-dependent Migration of Intestinal Epithelial Cells

Inflammatory bowel diseases are associated with increased risk of developing colon cancer. A possible role of the pro-inflammatory leukotriene D4 (LTD4) in this process has been implicated by the findings that LTD4 can signal increased proliferation and survival, both hallmarks of a cancer cell, in non-transformed intestinal epithelial cells. Here we make the novel finding that LTD4 can also signal increased motility in these cells. In parallel, we found that LTD4 induced a simultaneous transient 10-fold increase in Rac but not Cdc42 activity. These data were also supported by the ability of LTD4 to activate the Rac GDP/GTP exchange factor Vav2. Further, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation, a possible upstream activator of the Vav2/Rac signaling pathway. The activation of Rac was blocked by the PI3K inhibitors LY294002 and wortmannin and by transfection of a kinase-negative mutant of PI3K or a dominant-negative form of Vav2. Furthermore, Rac was found to co-localize with actin in LTD4-generated membrane ruffles that were formed by a PI3K-dependent mechanism. In accordance, the inhibition of the PI3K and Rac signaling pathway also blocked the LTD4-induced migration of the intestinal cells. The present data reveal that an inflammatory mediator such as LTD4 cannot only increase proliferation and survival of non-transformed intestinal epithelial cells but also, via a PI3K/Rac signaling pathway, trigger a motile response in such cells. These data demonstrate the capacity of inflammatory mediators to participate in the process by which inflammatory bowel conditions increase the risk for colon cancer development.

Effect of acetylation on conformation and bilirubin-binding properties of goat serum albumin

Four acetylated derivatives of goat serum albumin with percentage modifications of 18, 40, 53 and 93% were checked for bilirubin-binding and conformational properties. Acetylation caused marked changes in protein conformation, as evidenced by double immunodiffusion and proteolytic digestion results, as well as a decrease in bilirubin binding. An increase in ionic strength had a dramatic effect on the bilirubin-binding characteristics of modified proteins. The results suggest that the lysine residues of goat serum albumin modified in this study are not involved in bilirubin-albumin interaction.

Involvement of lysine residues of goat serum albumin in high-affinity binding of bilirubin

Four maleylated derivatives of goat serum albumin having percent modification as 40%, 46%, 84% and 98% were prepared using varying molar ratio of maleic anhydride over protein. These preparations were found to be pure, both with respect to size and charge as judged by gel filtration and polyacrylamide gel electrophoresis. Maleylation caused significant change in protein conformation as revealed by the change in Stokes radius and frictional ratio of serum albumin, from 3.46 nm and 1.28 to 4.96 nm and 1.79, respectively, upon 98% modification. Immunodiffusion results of native and modified albumins with anti-goat serum albumin antiserum also suggested significant conformational changes in serum albumin upon maleylation. About 88% reduction in bilirubin binding was observed after modification of 98% amino groups of serum albumin as studied by visible difference spectroscopy at pH 8.0, and at 0.15 ionic strength. Increase in ionic strength to 1.0 did not lead to any significant reversal in bilirubin binding. These results prove the involvement of lysine residues in bilirubin-albumin interaction.

Probing the determinants of protein solubility with amino acid modification.

Chemical modification was used as a probe to study the effect of structural features of serum albumin (charge, conformation, surface hydrophobicity, etc.) on its solubility behavior in concentrated ammonium sulfate solutions. Four different acetylated derivatives of goat serum albumin namely 18% acetylated, 40% acetylated, 53% acetylated, and 93% acetylated albumins were prepared. The homogeneity of these preparations was established by gel chromatography and polyacrylamide gel electrophoresis. Hydrodynamic data on the Stokes radius of native and acetylated albumins suggested gradual change in conformation on increasing modification. Solubility experiments performed in concentrated ammonium sulfate solutions at pH 7.0 and at 30 degrees C showed a slight decrease in salting-out parameter, Ks, up to 40% modification, whereas a significant decrease was obtained at higher modification. However, the salting-out parameter, beta, decreased monotonously. Similar decrease in these parameters was also observed with different modified albumins at other pH values viz. pH 5.5, 4.5, and 3.6. From these results we conclude that the decrease in solubility of serum albumin on increasing modification was primarily due to change in conformation.

Avoidance of endotoxin-induced inflammation during studies of albumin clearance from caprine joints.

Intra-articular injection of radiolabelled, commercial goat serum albumin (GSA) produces acute arthritis in caprine joints. This inflammation distorts clearance values and vitiates studies of normal lymphatic function. Endotoxin, routinely found in commercial albumin preparations, appears to cause this local reaction. We describe a simple method for the preparation of low-endotoxin, radioiodinated serum albumin from aseptically collected serum. We have used this technique to prepare GSA for use as a tracer molecule in clearance studies of synovial joint lymphatic function. The isolated protein exhibits antigenic and chemical characteristics indistinguishable from those of commercial GSA but contains at least 1000-fold less endotoxin. In contrast to commercial GSA preparations, this albumin does not produce local inflammation when injected into synovial joints and is cleared from caprine joints in the normal monoexponential manner. Low levels of endotoxin seriously distort studies of articular albumin kinetics and may induce comparable artifacts when commercial protein preparations are used to study other physiological systems.