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In-gel detection of esterase-like albumin activity: Characterization of esterase-free sera albumin and its putative role as non-invasive biomarker of hepatic fibrosis
⁎Corresponding author at: Biochemical and Clinical Genetics Laboratory, Section of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh 202002, UP, India. Tel.: +91 571 270092x3445. ahmadriaz2013@gmail.com (Riaz Ahmad)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Albumin is a globular and un-glycosylated multifunctional plasma protein and thus correlated with several human diseases. Owing to esterase contamination, albumin levels are usually misleading. In this study, we propose methodical accuracy for albumin estimation taking healthy and fibrotic rats. Liver fibrosis in rats was generated by N′-Nitrosodimethylamine (NDMA) (10 mg/kg body weight) within three weeks followed by its confirmation through H&E and immunohistochemical staining for α-SMA expression. Animal sera were screened by native polyacrylamide gel electrophoresis (native-PAGE) (7.5%). In-gel esterase-like albumin activity was detected using α- and β-naphthyl acetate (5.58 × 10−3 mM; pH 7.5) as substrate. Sera albumin was purified from unstained PA gel-slices through electroelution. Subsequent to conformation of albumin purity by its molecular weight determination using SDS–PAGE (10%) and peptide mass fingerprinting by MALDI-TOF-MS, samples were treated with different concentrations of urea. Urea-treated albumins were screened for esterase activity, conformational change and, albumin levels by immunoblotting. Our results demonstrate that esterase-like albumin activity in rat sera albumin is located in domain-III. The esterase-like activity remains detectable up to 4 M urea, which diminishes with increasing urea concentrations. Further, immunoblotting of urea-treated albumin samples displays a significant decline in purified protein bands, indicating hypoalbuminemia during hepatic fibrosis in rats. In conclusion, the present approach of albumin separation and estimation is of potential interest and may be recommended for diagnostic purposes.
Keywords
Albumin
Electrophoresis
Esterase-like albumin activity
Hepatic fibrosis
N′-Nitrosodimethylamine
Urea denaturation
1 Introduction
Albumin is a 65–67 kD globular, un-glycosylated and the most abundant multifunctional plasma protein in mammals. It is involved in many vital physiological functions like maintenance of plasma oncotic pressure and pH and, regulation of the transport of fluids across the capillary membrane etc. Besides, the protein is a major carrier of endogenous molecules, metabolites, vitamins, fatty acids, bilirubin, drugs and other xenobiotics into the blood. Moreover, albumin has been reported as the most important antioxidant substance in plasma (Roche et al., 2008).
Published literature on human serum albumin (HSA) suggests that it contains at least two distinct binding sites; site-I for the binding of bulky heterocyclic molecule with centrally located negative charge (like a large number of drugs) and site-II, which binds aromatic carboxylic acids with hydrophobic centers (Sudlow et al., 1975, 1976). An interesting attribute of HSA is that it also possess esterase-like activity with p-nitrophenol (Means and Bender, 1975), α- & β-naphthyl acetate (Casida and Augustinsson, 1959; Morikawa et al., 1979; Ahmad et al., 2012), nicotinate esters (Salvi et al., 1997), aspirin (Rainsford et al., 1980), ketoprofen glucuronide (Dubois-Presle et al., 1995), carprofen acylglucuronide (Georges et al., 2000), cyclophosphamide (Kwon et al., 1987) and a large number of long and short chain fatty acid esters (Tove, 1962). This esterase-like activity of sera albumin has been endorsed to the presence of arginine and tyrosine residues at 410 and 411 positions (Watanabe et al., 2000). This has been confirmed that esterase-like activity of sera albumin is inhibited by various drugs and actually the acetylation of lysine residues results in the inhibition of esterase-like activity of sera albumin (Lockridge et al., 2008).
Structural and functional impairments in albumin are attributed to various pathophysiological conditions like diabetes (Doweiko and Bistrain, 1994), osteoarthritis (Ahmad et al., 2011a) and advanced liver diseases (Zoli et al., 1991; Sugimura et al., 1994). Due to esterase–albumin complex formation in these pathologies, use of albumin as non-invasive marker may be misleading. However, in many of the liver diseases plasma levels of albumin are reported to decline due to its reduced synthesis and oxidative modifications that result in its altered binding with bilirubin (Masood et al., 2002). One of the major limitations to the clinical use of direct markers of liver fibrosis is that they are not routinely same in all settings. As a result, simple and less expensive/cheap markers are needed to be used more extensively in the clinical practice.
A reliable marker for hepatic fibrosis must fulfill the following criteria: (a) ability to quantify total mass of fibrous tissue in the liver; (b) ability to evaluate whether the liver is in a pro-fibrogenic or anti-fibrogenic state and, (c) be sensitive enough to determine the response of the liver to the treatments designed to combat fibrosis. Like many other pathologies, there has been a debate on albumin levels in liver fibrosis also, where several researchers have reported an increase as well as decrease in the levels of albumin during liver fibrosis (Rothschild et al., 1969; Panduro et al., 1990; Masood et al., 2002; Sakaida et al., 2004; George, 2006). The discrepancy in quantifying the exact levels of albumin may be attributed to esterase–albumin complex and the sensitivity of site-I and -II to bind with various molecules. Although there are other methods available for the detection of liver fibrosis such as liver biopsy examinations, transient elastography and several laboratory tests, but still exists scarcity of non-invasive supportive biomarker. Therefore, discovery of non-invasive biomarkers to detect liver fibrosis shall be a priority. In the present study, we demonstrate a methodical procedure for albumin separation and purification free from esterase contamination in a mammalian model of liver fibrosis utilizing the following procedural steps: (1) In-gel detection of esterase-like albumin activity, (2) purification and further confirmation of rat sera albumin by electroelution and SDS–PAGE followed by MS analysis respectively, (3) inhibition of esterase-like activity of purified albumin by urea and, (4) confirmation of sera albumin by spectroscopy and later by immunoblotting to show its accurate levels in healthy and fibrotic rats.
2 Materials and methods
2.1 Chemical
Acrylamide, bis-acrylamide, ammonium persulfate (APS), TEMED, and N′-Nitrosodimethylamine (NDMA), were procured from Sigma–Aldrich. α- and β-naphthyl acetate, urea and Tris buffer were purchased from SRL, India, goat anti-mouse IgG-HRP conjugated antibody was purchased from CALTAG Laboratories, Bangkok. α-smooth muscle actin (α-SMA) antibodies were obtained from Trend Bio-products Pvt. Ltd., India. All the other chemicals and reagents used were of analytical grade.
2.2 Care and maintenance of animals
Healthy adult male albino rats Rattus norvegicus of Wistar strain, weighing around 145 ± 10 g were used in the present study. The animals were housed in well aerated polycarbonate cages (12 h: 12 h = light: dark period) with proper humane care and were fed regularly with commercial, sterilized diet (Ashirwad Industries Pvt. Ltd., Mohali, Punjab, India) and water available ad libitum. They were acclimatized for a week before taking them for the treatment. All the experiments were performed according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.
2.3 Induction of hepatic fibrosis
Fibrosis was induced essentially as described previously (Ahmad et al., 2009). Briefly, the animals were divided into two groups, comprising of fifteen rats each. One group received intraperitoneal injections of NDMA (diluted with 0.15 M NaCl) in doses of 10 mg kg−1 body weight and the other group, serving as control, received normal saline 0.15 M NaCl in doses of 10 mL kg−1 i.p. The injections were given on three consecutive days of each week for three weeks, as described earlier (Ahmad et al., 2009). Five animals from each group were anesthetized and sacrificed weekly on days 7, 14 and 21 from the start of treatment.
2.4 Histopathological and immunohistochemical analyses of liver
Livers from sacrificed rats of each category were quickly excised with sterilized scissors and forceps, washed with phosphate buffered saline (PBS) to remove other tissue debris. A piece of liver tissue (from each animal) was fixed in formalin (10%) and processed for histopathological studies. The degree of hepatic fibrosis was monitored by Hematoxylin and Eosin (H&E) staining and immunohistochemical localization of α-smooth muscle actin in paraffin-embedded 5 μm thick serial liver sections. Immunohistochemistry was carried out as described in our previous reports (Ahmad et al., 2009). Stained slides were examined and photographed under Nikon microscope with an LCD attachment (Model: 80i).
2.5 Collection of sera and protein estimation
Blood was collected through cardiac puncture directly on weekly basis i.e. on days 7, 14 and 21 from both treated as well as control groups, from the beginning of experiment. Blood samples were kept standing at RT for about an hour to ooze out sera. Samples were centrifuged at 3000 rpm and 4 °C for 8–10 min and the resulting clear, pale yellow colored sera were either analyzed afresh or stored in aliquots at −20 °C for further biochemical investigation.
Protein concentration in the sera samples was determined by following the protocol of Lowry et al. (1951) using Folin phenol as coloring reagent and taking bovine serum albumin (BSA) as the standard. Absorbance was taken at 660 nm on a 10 UV–Visible GeneSys spectrophotometer.
2.6 In-gel staining of esterases
Vertical slab, non-denaturing 7.5% polyacrylamide gel (100 × 80 × 1 mm) electrophoresis of collected sera samples was carried out according to the protocol of Laemmli (1970), with the modification that samples, gels and running buffers were lacking SDS. The gels contained acrylamide (Acrylamide: bis-acrylamide = 29.2:0.8) and 10% glycerol. Equal quantities of protein (8 μg) were loaded in each well and gels (in triplicate) were run in buffer containing Tris (24 mM) and glycine (194 mM) at room temperature. Following completion of electrophoretic runs, one of the gels was stained with Coomassie Brilliant Blue R-250 (CBB-R250) and destained overnight with 7% (v/v) glacial acetic acid. The gel, processed for esterase localization was incubated in a reaction mixture containing α, β-naphthyl acetate (5.58 × 10−3 mM, pH 7.5) as substrates in the presence of Fast Blue RR at 25 °C (Ahmad et al., 2012). Upon development of the dark brown bands, indicating esterase activity, the reaction was stopped by fixing the gel in 7% (v/v) glacial acetic acid for 20 min, followed by preservation of the gel in 5% (v/v) acetic acid prepared in 10% methanol.
2.7 Purification of sera albumin by electroelution
The zones (in the unstained gel), corresponding to albumin bands in CBBR stained gels were cut and placed in Tris–glycine (24 and 194 mM, respectively) as equilibration buffer at −20 °C. Albumin purification was carried out by electroelution in Tris–glycine buffer of the same molarity at 4 °C for 10 h. Briefly, for electroelution, gel pieces corresponding to the stacking zone of albumin activity were excised in small pieces and kept in dialysis tubing containing Tris–glycine buffer. Dialysis bags, partially submerged in running buffer, were placed in a horizontal electrophoretic assembly under the influence of electric field. Horizontal flow of gel pieces in the tubing was prevented by clamping in the middle of the dialysis tubing. The current was allowed to flow for 10 h at 4 °C (based on our pilot experiments) for complete elution of albumin from the gel slices. The technique of electroelution is a routine and established procedure in our laboratory.
2.8 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of purified albumin
The albumin, thus purified, was confirmed on 10% SDS–PAGE essentially carried out according to the protocol of Laemmli (1970). Silver staining of the gels was carried out following the procedure of Nesterenko et al. (1994). Freshly prepared chicken natural actomyosin (NAM) was used as the molecular weight marker.
2.9 Albumin confirmation by MALDI-TOF/TOF MS analysis
The protein band of interest (pAlb) was excised, diced into small pieces and destained. The destaining solution was then removed and the gel pieces were completely dried using acetonitrile and Speedvac and reduced with 10 mM DTT (1 h at 56 °C), alkylated with 55 mM iodoacetamide (45 min at room temperature in the dark). In-gel trypsin (12.5 ng μL−1 in 50 mM NH4HCO3 and 5 mM CaCl2) digestion was carried out at 37 °C overnight. The extraction of peptides after in-gel digestion was performed with 50% acetonitrile and 0.1% trifluoroacetic acid (TFA) and peptides obtained were mixed with α-cyano-4-hydroxycinnamic acid (HCCA) matrix. MALDI-TOF/TOF MS analysis was performed with MALDI-TOF/TOF MS, Bruker Daltonics, Ultraflex III instrument. Further analysis was carried out with Flex Analysis Software for obtaining the peptide mass fingerprint (PMF). PMF and peptide fragments were examined with the MASCOT program (www.matrixscience.com) using NCBInr/Swiss-Prot sequence database.
2.10 Urea denaturation of purified albumin
The albumin purified from different treatment groups was incubated with different urea concentrations i.e. 1, 2, 3, 3.5, 4, 4.5 and 5 M at room temperature for 12 h. Urea treated albumin samples were analyzed through native-PAGE (run in duplicate). The gels were stained with CBB-R250 and replica gel was incubated in reaction mixture containing α-naphthyl acetate and β-naphthyl acetate as substrates in the presence of Fast Blue RR at 25 °C (Ahmad et al., 2012). Once the optimum visibility of bands was achieved, the reaction was stopped by transferring the gels into 7% (v/v) glacial acetic acid.
2.11 Spectral analysis of urea-treated albumin samples
Purified albumin (from all treatment categories) was treated with variable concentrations of urea and incubated at room temperature for 12 h. Spectral analysis of these samples was carried out in the range of 250–300 nm. Tris–glycine, in addition to varying concentrations of urea i.e. 1, 2, 3, 3.5, 4, 4.5 and 5 M was used as the reference solution.
2.12 Albumin detection by Western immunoblotting
Western immunoblotting was performed according to the methodology followed by Hasnain et al. (2004). Electrophoretic runs of SDS-PA gels were equilibrated in Tris–glycine transfer buffer (24 mM Tris, 194 mM glycine and 10% v/v methanol) for 25–30 min. The electrotransfer was carried out on PVDF membranes (0.45 mm, BioRad, USA) at 125 V/200 mA for 2 h at 4 °C. Subsequently, the membranes were washed thrice in PBS (50 mM, pH 7.1) and treated with blotto (5% w/v non-fat dry milk in PBS) for 1 h at room temperature. All the remaining steps of incubation and washing were furnished in the same blocking solution (i.e. blotto) containing 0.1% Tween-20 (PBS-T) (w/v). The blot was then incubated for 2 h in primary antibody (rabbit anti-rat albumin antisera) diluted to 1 mg/mlwith gentle shaking and, washed for 30 min with 3 changes of PBS-T. The membrane was then treated with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG immunoglobulins) diluted × 200 for 1 h at RT. Subsequently, the membrane was washed thrice with PBS-T to remove the unbound antibody. Chemiluminescence detection of peroxidase activity was performed with LumiGLo Chemiluminescent kit (KPA, USA) wherein the membrane was incubated with luminol reagents, exposed to sensitive USG films for ∼5 min under the dark, and developed.
2.13 Statistical analysis
The values of control and treated specimens were compared and presented as mean ± SD. To test the significant differences among the obtained values, Student’s t-test was applied at P < 0.05.
3 Results
Hepatic fibrosis is a clinical condition resulting in greatly hampered liver functioning, due to abnormal accumulation of extracellular matrix proteins, in response to acute or chronic hepatic injury. Variety of causes contributing to liver injury includes viral infections, drugs, autoimmune, cholestasis and some metabolic disorders (Ahmad and Ahmad, 2012). In the present study, we have induced hepatic fibrosis in rats by N′-Nitrosodimethylamine (NDMA) and selected it as a disease model to compare the obtained results of healthy with fibrotic animals. The fibrosis thus generated perfectly imitates the hepatic fibrosis in humans.
3.1 Histopathology and immunohistochemical staining of liver biopsies
Hematoxylin and Eosin stained liver biopsies demonstrate disruption in lobular architecture of the liver with severe neutrophilic infiltration and patches of necrosis in the liver (Fig. 1A and B). Confirmation of the diseases was done through immunohistochemical staining of the liver biopsies for α-smooth muscle actin (α-SMA) localization, the diagnostic membrane marker of activated hepatic stellate cells (HSCs). Liver section showed numerous α-SMA positive HSCs (activated) in the necrotic region of NDMA-induced fibrotic liver (Fig. 1C and D).
Histopathology of liver sections. Histological confirmation of NDMA-induced hepatic fibrosis in rats. Hematoxylin and eosin staining (A and B) of liver sections. (A) Control liver (10×) showing normal architecture, (B) Day-21 of NDMA treatment showing severe hemorrhagic necrosis, congestion, lymphocyte infiltration (20×). Immunohistochemical staining of α-smooth muscle actin (α-SMA) (C and D) during the pathogenesis of NDMA induced hepatic fibrosis (C) control liver (10×) showing absence of α-SMA staining. (D) Intense staining of α-SMA demonstrating widespread activation of hepatic stellate cells in the fibrotic zone (10×).
3.2 Electrophoretic profiling and identification of major sera fractions
Electrophoretic profiling of the sera of control and fibrotic rats under non-denatured conditions showed almost all the major sera protein fractions, with a thick band of albumin toward the anodal region (Fig. 2A).![Electrophoretic studies of rat sera. Native polyacrylamide gel electrophoretic (PAGE) profiles of control (C) and fibrotic (F) rat sera samples [A] CBBR staining [B] Esterase staining. SDS–PAGE profiles of control (C) rat sera and purified albumin samples (pAlb). [C] CBBR staining, [D] Esterase staining. [E] Gel portion showing esterase-like albumin activity zone in control and fibrotic animals and their relative intensity.](/content/184/2017/10/5/img/10.1016_j.arabjc.2014.10.016-fig2.png)
Electrophoretic studies of rat sera. Native polyacrylamide gel electrophoretic (PAGE) profiles of control (C) and fibrotic (F) rat sera samples [A] CBBR staining [B] Esterase staining. SDS–PAGE profiles of control (C) rat sera and purified albumin samples (pAlb). [C] CBBR staining, [D] Esterase staining. [E] Gel portion showing esterase-like albumin activity zone in control and fibrotic animals and their relative intensity.
3.3 In-gel detection of esterase-like albumin activity
Esterase-like albumin activity was observed in the control and fibrotic sera samples under native conditions when gels were incubated with α-, and β-naphthyl acetate at room temperature. A thick band, corresponding to the stacking zone of albumin in CBB stained gels, was observed indicating esterase-like albumin activity. Few low intensity minor bands of esterase activity were also detected in the upper region of the gels (Fig. 2B).
3.4 Studies on albumin purified through electroelution
Screening of the electroeluted albumin samples from sera of fibrotic and control rats was carried out under non-denaturing and denaturing conditions to confirm its purity. CBB stained native gels demonstrate the presence of a single band corresponding to albumin activity, while the same activity with single band was also observed in substrate specific staining of esterases suggesting that albumin retains its esterase-like activity even after purification (Fig. 2C and E). Electrophoretic profiles under denatured conditions support the presence of a single band in the electroeluted samples. GelPro analysis reveals ∼66 kD band stacked corresponding to albumin in SDS-PA gels (Fig. 3A). The purity of albumin, thus purified by electroelution, was analyzed and confirmed for rat serum albumin by mass spectrometric peptide mass fingerprint. MALDI-TOF/MS spectrum derived from a representative peptide is shown in Fig. 3B.![Characterization of purified albumin. [A] SDS–PAGE Profile: M, MW marker (chicken actomyosin); C, control sera samples; F, sera from fibrotic animal; pAlb-C, purified albumin from control rat and pAlb-F, purified albumin from fibrotic animal. [B] peptide mass fingerprint (PMF)/MS spectrum generated by tryptic digestion of rat serum albumin.](/content/184/2017/10/5/img/10.1016_j.arabjc.2014.10.016-fig3.png)
Characterization of purified albumin. [A] SDS–PAGE Profile: M, MW marker (chicken actomyosin); C, control sera samples; F, sera from fibrotic animal; pAlb-C, purified albumin from control rat and pAlb-F, purified albumin from fibrotic animal. [B] peptide mass fingerprint (PMF)/MS spectrum generated by tryptic digestion of rat serum albumin.
3.5 Effect of urea on sera esterase-like albumin activity
Albumin thus purified was treated with 1.0, 2.0, 3.0, 3.5, 4.0, 4.5 and 5.0 M urea concentration. The treated samples were run on polyacrylamide gels and stained for esterase activity. The results showed the presence of esterase-like albumin activity in samples treated with urea up to 4 M concentration (Fig. 4A), which subsequently disappears from 4.5 M urea treatment and onward (Fig. 4B). Conformational changes in sera albumin at 3, 4 and 5 M of urea in control and fibrotic rats are shown in Fig. 5A and B, respectively. A distinct peak at ∼272 nm was observed that essentially shows perturbation range of tyrosine residues. Compared to the control, spectra of urea- treated albumin samples of fibrotic rat showed lower ΔA values and no shift in spectral peak (Fig. 5A and B). Immunoblotting data on the urea-treated albumin samples from control and progressive fibrotic stages clearly display a significant decline in the intensity of purified bands, indicative of hypoalbuminemia during hepatic fibrosis in rats (Fig. 5C).![Urea denaturation of purified albumin. Native PAGE profile of urea-denatured purified albumin. Control rat sera (1); Purified albumin from fibrotic animal with increasing molarities of Urea: [A] (2) 1 M, (3) 2 M, (4) 3 M, (5) 3.5 M, (6) 4 M; [B] (2) 4.5 M and (3) 5 M urea concentration.](/content/184/2017/10/5/img/10.1016_j.arabjc.2014.10.016-fig4.png)
Urea denaturation of purified albumin. Native PAGE profile of urea-denatured purified albumin. Control rat sera (1); Purified albumin from fibrotic animal with increasing molarities of Urea: [A] (2) 1 M, (3) 2 M, (4) 3 M, (5) 3.5 M, (6) 4 M; [B] (2) 4.5 M and (3) 5 M urea concentration.
![Characterization of albumin during hepatic fibrosis. UV difference spectra of purified albumin between 250 and 300 nm. [A] Albumin purified from control/untreated rat sera, [B] Albumin purified from fibrotic rat sera. [C] Immunoblots of purified albumin in control and NDMA treated samples of rat. Bar graph showing quantitative variations in the expression of albumin protein obtained during Western immunoblots.](/content/184/2017/10/5/img/10.1016_j.arabjc.2014.10.016-fig5.png)
Characterization of albumin during hepatic fibrosis. UV difference spectra of purified albumin between 250 and 300 nm. [A] Albumin purified from control/untreated rat sera, [B] Albumin purified from fibrotic rat sera. [C] Immunoblots of purified albumin in control and NDMA treated samples of rat. Bar graph showing quantitative variations in the expression of albumin protein obtained during Western immunoblots.
4 Discussion
Serum, an important component of blood, lacks blood cells (red or white), clotting factors and fibrinogens. It comprises immunoglobulins, antigens, hormones, electrolytes along with other exogenous substances. It is regarded as the best source for proteomic analyses and hence, used in various diagnostic tests as a non-invasive source in many pathophysiological conditions (Adkins et al., 2002; Ahmad et al., 2008; Madian and Regnier, 2010). The major protein in the serum, constituting about 50%, is albumin, a globular protein of nearly 65–67 kD (Hasnain et al., 2004; Ahmad et al., 2007, 2011a). Albumin is synthesized in the liver as a precursor- preproalbumin and with N-terminal peptide, which during post-translational processing of the nascent protein is removed in the rough endoplasmic reticulum. Subsequent to cleavage of proalbumin in Golgi vesicles, albumin is secreted out. That albumin exhibits esterase-like activity has been a matter of great interest due to the potential of albumin as a candidate biomarker in several diseases (Dubois-Presle et al., 1995; Salvi et al., 1997; Sakurai et al., 2004). It is already established that esterase-like albumin activity of human serum albumin (HSA) is attributed to sub-domain IIIA (Watanabe et al., 2000). NMR spectroscopy data and site-directed mutagenesis studies revealed significantly augmented rates of hydrolysis of aspirin in the presence of HSA, thereby confirming the esterase-like activity of HSA (at site I) and the role of Arg-410 and Tyr-411 for the esterase activity of HSA (Hawkins et al., 1969; Burch and Blazer-Yost, 1981; Honma and Ishikawa, 1991; Watanabe et al., 2000). Since serum contains adequate amount of esterases which are involved in drug metabolism (Hubbard et al., 2008; Ahmad et al., 2012), their levels along with esterase-like albumin activity have been employed as prognostic markers for various diseases (Aoyagi et al., 1984; Ahmad et al., 2011a, 2011b, 2012; Thangthaeng et al., 2011). In the present study, we analyzed the major sera protein fractions, in-gel histochemical staining for esterase-like albumin activity, and purification of serum albumin (from all treatment groups) by electroelution followed by its molecular weight estimation and UV-spectroscopy. Further we have analyzed the albumin activity in urea-treated protein samples of control and fibrotic rats by immunoblotting.
N′-Nitrosodimethyl amine-induced hepatic fibrosis in rats, an established model in our laboratory (Ahmad et al., 2009, 2012, 2014; Ahmad and Ahmad, 2014), was confirmed by H&E staining along with the immunohistochemical staining of α-smooth muscle actin (diagnostic marker of hepatic stellate cell activation) (Fig. 1). We have used the fibrotic rat model to authenticate our approach of albumin characterization during diseased state. The protein types (s), in their normal conformation, were identified by native gels run under non-denaturing conditions. Since native gel electrophoresis does not use a charged denaturant, the proteins being separated experience different electrophoretic forces dependent on the shape of the overall structure and may be visualized by general protein staining as well as by substrate specific (enzyme) staining (Ahmad et al., 2008, 2011b). Our results demonstrate at least five major activity zones of varying intensity in control and fibrotic animals, namely: globulins, haptoglobins, transferrins, albumin and prealbumin along with the identification of esterase-like albumin activity in the whole sera of rats (Fig. 2A and B).
The electroeluted and purified albumin resolved as single band on SDS–PAGE also shows almost comparable esterase-like albumin activity on native gels in control and fibrotic rats (Fig. 2C). Therefore, the present approach provides a useful and convenient method for studying oxidative modification and a variety of post-translational modifications of sera albumin.
Esterase-like activity of albumin can be visualized by substrate staining with α-, and β-naphthyl acetate in the presence of Fast Blue RR salt as a dye coupler (Ahmad et al., 2012). Albumin preparations have been found to hydrolyze many xenobiotic esters. The term ‘esterase-like’ activity of albumin has been introduced to explain these observations. In some cases however, it is still not clear if this activity is intrinsic to the albumin molecule or is in fact due to contamination of esterase impurity in the albumin preparation. Chapuis et al. (2001) had previously demonstrated that esterase-like albumin activity in humans is due to contamination of low amounts of other esterases. In our conditions, the esterase-like albumin activity was detected with almost equal intensity in control as well as fibrotic animals (Fig. 2C), signifying the sensitivity of the native PAGE. During electrophoretic separation of serum proteins under native conditions, albumin, due to its net negative charge, migrates toward the anode. The present system may offer separation of proteins with higher electrophoretic mobility than albumin in relatively more concentrated gels (with smaller pore size) and may prove suitable for separation of multiplex proteins also (Yan et al., 2007). Further our SDS–PAGE results of the purified albumin demonstrate the presence of single band of albumin that stacks corresponding to Mr ∼66 kD, in control as well as fibrotic sera samples (Fig. 3A). Our MS results (Fig. 3B) are also indicative of purity of the preparation (albumin) and the sensitivity of the technique adopted.
Urea is the most popular denaturant, being used extensively in biochemistry, not only to denature protein at higher concentrations, but also to promote controlled folding. Perhaps the most widely studied model of globular proteins is serum albumin, synthesized by the liver, accounting for 60% of the total globular proteins in blood plasma and constituted by around 585 amino acid residues, whereas its secondary structure is constituted by 67% alpha helix and 17 disulfide bridges that confer to the protein a relatively strong stability (Carter and Ho, 1994). An interesting study by Itri et al. (2004) shows that the presence of 3 M urea induces folding in BSA (revealed by SAXS) and its secondary structure remains almost unaltered in terms of helicity as evidenced by CD data. On the other hand, 5 M urea showed pronounced effect on protein structure: the α-helical content suffers a remarkable decrease accompanied by a significant change in protein conformational as revealed by the values observed for macromolecule radius of gyration (Rg = 72 Å) and maximum dimension (Dmax = 240 Å) as well as from functional behavior. Moreover, the above cited study also demonstrate that addition of 3 and 5 M urea in equal concentration of BSA, slightly decreases the tryptophan emission, suggesting that urea unfolds the protein in the interface. Gull et al. (2007) reports that in case of urea-induced unfolding of HSA, domain III is primarily responsible for intermediate formation, wherein domains I and III unfold before 5 M and domain II unfolds beyond it. Moreover, the presence of a single cysteine, tryptophan and tyrosine residue in domains I, II and III respectively, offers a chance to pursue the unfolding and refolding studies. Subdomain IIIA of HSA possesses a well known esterase-like activity toward substrates such as p-nitrophenyl acetate and several N-carbobenzoxy-d(l)-alanine p-nitrophenyl esters (Watanabe et al., 2000).
Our results based on electrophoretic profiling and spectroscopy data on urea induced changes in esterase-like albumin activity are in conformity with the above cited reports and clearly demonstrate that at >4 M urea concentrations esterase-like albumin activity is completely abolished (Fig. 4A and B). The present results, however, partially contradict the findings of Thangthaeng et al. (2011), wherein esterase-like albumin activity was not detectable in the samples with ⩾2 M urea concentrations. It is intriguing that in our case, similar to HSA, the esterase like albumin activity in rat sera albumin is located in domain-III, which is detectable apparently due to unfolding of protein up to 4 M urea and later, diminishes on increasing the urea concentration (Fig. 5A and B). Now it is evident that rat serum albumin obtained from the control and fibrotic rats shows resistance up to 4 M urea-treatment implying the stability and sensitivity of esterase binding domain of albumin at ⩽4 M. Moreover, immuno blotting (Fig. 5C) of 4.5 M treated albumin sample reveals a significant decline in sera albumin concentration or hypoalbuminemia during progressive hepatic fibrosis.
5 Concluding remarks
Since sera albumin is taken as a non-invasive biomarker in most of the diseases, its characterization is essential in those conditions where there is an apparent controversy on albumin levels. In this communication, we demonstrate a methodical approach of albumin purification and its subsequent characterization using various techniques such as MALDI-TOF/MS, spectroscopy and Western immunoblotting. It is concluded that the inhibition of esterase-like albumin activity by urea-treatment may be a potential approach for accurate albumin estimation particularly in cases where probability of esterase contamination is high. Furthermore, purification and characterization of albumin during hepatic fibrosis suggest hypoalbuminemia and supports its putative role for diagnostic purposes in addition to other non-invasive disease biomarkers.
Acknowledgements
The authors sincerely acknowledge the financial support from the Department of Science and Technology (DST), New Delhi, India, in the form of DST-INSPIRE fellowship to AA. Thanks are also due to the Chairman, Department of Zoology, Aligarh Muslim University, for necessary laboratory facilities. Outsourcing for MALDI-MS analysis was done from Sandoor Proteomics, Hyderabad, India.
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