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. 2020 Jul 20;10(8):343. doi: 10.1007/s13205-020-02338-7

A solvent-free delipidation method for functional validation of lipases

Achintya Kumar Dolui 1,2, Panneerselvam Vijayaraj 1,2,✉
PMCID: PMC7371773  PMID: 32714738

Abstract

Extracting protein in its active form is critical for its functional characterization, and lipid removal is an essential step in the protein extraction process for further downstream applications. In the present study, we revisited the delipidation protocol and developed a rapid, solvent-free delipidation method using activated silica. The delipidated samples showed improved optical clarity and a significant reduction of endogenous lipids. The functional integrity of the lipases present in the delipidated sample was validated by in vitro enzyme assay using physiological substrate which includes neutral lipid as well as phospholipid. The accessibility of active site of the extracted enzymes was demonstrated by activity-based protein profiling (ABPP), a functional chemoproteomic approach. Detection of serine hydrolases using ABPP probe labeling was enhanced upon delipidation. Further, the total polyphenol content was significantly reduced, which helps to enhance the protein enrichment and small-molecule inhibitor screening by ABPP. Collectively, these results suggest that the present solvent-free delipidation approach is efficient and highly compatible with the functional characterization of the enzymes, particularly lipid hydrolases.

Keywords: Delipidation, Enzymes, Lipases, Rice bran, Lipids, Activity-based protein profiling

Introduction

Identification and functional characterization of the lipid metabolizing enzymes particularly lipases have a vital role in the food and pharmaceutical industries. Lipases catalyze the hydrolysis of ester bonds of glycerolipids to free fatty acids (FFA). Lipases belong to the serine hydrolase family and share a similar catalytic triad (Ser–Asp–Glu/His) with serine proteases (Long and Cravatt 2011). Inhibition of lipases such as pancreatic lipase, monoacylglycerol lipase, and α/β hydrolase fold domain (ABHD) containing proteins is the therapeutic target for obesity and type II diabetes (Bachovchin and Cravatt 2012). Identification of inhibitor molecules against these enzymes is a promising strategy to prevent lipid metabolic disorders and cancer (Bachovchin and Cravatt 2012). In plants, functional characterization and stabilization or inhibition of lipases have shown a positive impact on the seed oil content (Morcillo et al. 2013; Kelly et al. 2013). Unfortunately, intrinsic lipids are the major challenge for both functional validation and screening of inhibitor molecules. The absolute quantification of lipase activity in the crude protein lysate using physiological substrate is difficult due to the interference from endogenous lipids (Jensen et al. 1983; Glamozzi et al. 2014). Although, many synthetic chromogenic substrates such as p-nitrophenyl derivatives, and fluorescent substrates were developed for measuring enzyme activity, they provide only a superficial insight into the activity. The utilization of radiolabelled substances mimics physiological substrates to some extent but has health concerns (Jensen et al. 1983; Stoytcheva et al. 2012). Further, interference of endogenous lipids was reported with accurate quantification and separation of proteins by SDS-PAGE (Eichberg and Mokrasch 1969; Sahu et al. 2017).

Traditionally, delipidation has been practiced using the solvents like chloroform, methanol, isopropanol, methyl-tert butyl ether and hexane for clarifying lipids from biological samples (Folch et al. 1957; Bligh and Dyer 1959; Ferraz et al. 2004; Hara and Radin 1978; Matyash et al. 2008). Although organic solvents are excellent delipidation agents, they have major drawbacks such as denaturation and precipitation of proteins (Cham and Knowles 1976). Also, the resolubilisation of the precipitated proteins is difficult (Vaisar 2009). Cham and Knowles (1976), reported the delipidation of serum without protein precipitation using butanol-di-isopropyl ether. This solvent-based delipidation might be suitable for non-enzymatic proteins; whereas, it is unsuitable for catalytic proteins due to the loss of structural integrity which leads to functional inactivity (Dowhan 1997; Onder et al. 2018). Alternatively, solvent-free delipidation methods have been reported using dextran sulphate, calcium chloride, α-cyclodextrin, Aerosil 380F and by high-speed centrifugation (Sharma et al. 1990; Dimeski and Jones 2011). Even the commercially available lipid removal agent known as Cleanascite used for serum samples has lower efficiency (Castro et al. 2000; Barrera et al. 2018). However, the functional state of the delipidated protein samples particularly enzyme activity has not been studied in detail.

The solvent-based delipidation interrupts the enzyme activity and protein solubilization, which makes the samples highly incompatible to functional proteomic analysis (Sajic et al. 2011). Among current proteome approaches to analyze enzyme activity, Activity-based protein profiling (ABPP) stands out as a powerful tool due to its multifunctional applications like discovering new enzymes, uncovering the differences in the enzyme activity during pathological states and screening of small-molecule inhibitors for the particular enzyme (Jessani and Cravatt 2004). Its a chemical proteomics approach that utilizes active site-directed small-molecule probes that covalently interact only with the active form of enzymes in native systems (Cravatt et al. 2008; Heal et al. 2011). The interactions can be detected by fluorescent gel imaging or mass spectrometry. The serine hydrolase probe has effectively been employed in the detection and identification of lipases as well as for screening the inhibitor molecules (Jessani and Cravatt 2004; Heal et al. 2011). The interference of lipids in the form of an endogenous substrate is a potential challenge in the ABPP assay due to the competitiveness between the serine hydrolase probes and endogenous lipids. Further, it weakens the interaction of the probe with the active site which leads to loss of protein detection by low signals (Glamozzi et al. 2014). Hence, preclearing or delipidation of the protein samples is mandatory for detecting lipases activity using physiological substrates or active site-mediated serine hydrolase probe. We developed a simple and efficient lipid removal protocol using activated silica which was validated with lipid-rich protein samples such as RB and mouse adipose tissue. The solvent-free delipidation protocol effectively removes the lipid components in the protein preparation for the functional proteome applications, and it is the first report on plant protein as well as on ABPP.

Materials and methods

Materials

The rice bran used in the study was received from Dr. A. Jayadeep, CSIR-CFTRI. The normal mice adipocyte tissues were obtained from the Animal House Facility with the approval of the IAEC (CPCSEA#49/PO/ReBi/S/1999/CPCSEA). ActivX™ TAMRA-FP Serine Hydrolase Probe was purchased from Thermo Fisher Scientific. The lipid substrates such as 1-2-dioleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine,1,2-dioleoyl-sn-glycero-3-phospho-1′-myo-inositol, 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dioleoyl-sn-glycero-3-phospho-1′-rac-glycerol were procured from Avanti Polar Lipids (Alabaster, AL). 1, 2, 3-Tri (cis-9-octadecenoyl)-glycerol and 1-Oleoyl-rac-glycerol were procured from Sigma-Aldrich. Silica gel (SiO2), Celite and other chemicals were procured from Sigma-Aldrich. Florisil PR grade, 60–100 mesh was procured from Supelco analytical. FFA fluorometric assay kit (700310) was procured from Cayman Chemical, USA.

Protein extraction

The protein extraction was performed by vortexing the bran with extraction buffer (50-mM Tris–HCl (pH 8.0), 150-mM NaCl, 1-mM MgCl2, 1-mM KCl, 10% Glycerol) for 30 min at 4 °C. The crude cell-free lysate was obtained by centrifugation at 5000 rpm for 10 min at 4 °C. Further, the soluble protein fraction was obtained by ultracentrifugation at 35,000 rpm for 90 min at 4 °C. After the centrifugation, the top lipid layer was removed and the supernatant was collected for delipidation treatment. For the mammalian sample, adipose tissues were collected from anesthetized mice and washed three times with cold PBS to avoid blood cells. The adipose tissues were minced into small pieces and protein was extracted by homogenization with the above-mentioned buffer and centrifuged at 5000 rpm for 10 min at 4 °C. The cell-free supernatant was used for delipidation.

Delipidation of protein samples

The solvent-free delipidation was performed in lipid-rich biological samples such as rice bran and adipose tissue using activated Silica (SiO2) gel (ASG). The silica gel was activated at 120 °C for 4 h and stored in a desiccator at room temperature. The amount of ASG and sample was calculated based on weight and volume (w/v), respectively. The preferred protein concentration of the samples was a minimum of 1 mg/mL. ASG was pre-equilibrated with a sample or extraction buffer to minimize the sample loss. The endogenous lipids were captured in ASG by mixing the protein sample using a rotatory mixer at 10 rpm at 4 °C for 30 min. Delipidation was tried at different ratios (w/v) of ASG and sample ranging from 1:2 to 1:10. After the incubation, the samples were centrifuged at 5000 rpm for 5 min, and the supernatant was collected for further study. Similarly, the delipidation of the rice bran sample was performed with other reported agents such as folorisil, and celite (1:1, w/v) for the comparative analysis. Protein estimation was performed for each delipidated sample.

Lipid analysis

The lipid analysis was carried out in both rice bran and mice adipose samples. The lipids were extracted from 100 μg of control (non-delipidated) or delipidated protein samples by Bligh and Dyer (1959) method and washed with 2% orthophosphoric acid. The nonpolar lipids were separated using petroleum ether: diethyl ether: acetic acid (70:30:1, v/v) and the phospholipids were separated using chloroform: methanol: ammonia (65:25:5, v/v) as the solvent system. The TLC plates were exposed to iodine vapors to visualize the lipids followed by MnCl2 charring.

Enzyme assay and lipid profiling

The functional integrity of the delipidated protein samples was monitored by enzyme assay, particularly for lipase activity (Dolui et al. 2020). The activity was measured by quantification of the released FFAs from the substrate using rice bran protein or adipose protein. The assay mixture consists of 50-mM Tris–HCl (pH 8.0), 1-mM MgCl2, 1-mM KCl, 10% Glycerol in the presence of 50μM substrate and 50 μg of protein (either control or delipidated as the enzyme source) in a total volume of 100 μL. The assay was performed at 37 °C for 30 min or different time intervals and stopped by the addition of 2:1 (v/v) chloroform and methanol. The lipids were extracted, and the products were visualized by resolving on a silica-TLC plate using the above-mentioned solvent systems. Further, the enzyme activity was quantified by estimating the FFA using a fluorometric estimation kit.

Quantification of fatty acid

The level of endogenous lipids or the efficiency of delipidation was monitored by quantifying the total fatty acid or FFA content using GC/MS analysis (Morrison and Smith 1964), and fluorescent‐based FFA estimation method as described by the manufacturer (Cayman Chemical, USA, Cat # 700310).

Activity-based protein profiling analysis

The functional integrity of the active site was monitored by a gel-based ABPP assay, particularly for serine hydrolase activity. The ABPP was performed for control as well as delipidated protein samples from rice bran and adipose tissue. The assay mixture consisted of 100-μg protein and 2-μM Active X-TAMRA serine hydrolase probe in the total volume of 50 μl using the above-mentioned extraction buffer. The reaction mixture was incubated for 60 min at 37 °C and terminated by the addition of 10-μL 4× loading buffer (200-mM Tris–HCl, pH 6.8, 400-mM DTT, 8% SDS, 0.04% bromophenol blue, and 40% glycerol) and boiled for 5 min. Proteins were resolved on a 12% (w/v) SDS-PAGE and the probe-labeled enzymes were detected under fluorescent scanning at 532 nm. Further, the gels were stained with coomassie brilliant blue R-250, and the images were documented.

Quantification of total phenolic content (TPC)

The level of TPC in the silica-treated rice bran and control samples was quantified by the Folin–Ciocalteu method using gallic acid (GA) as standard (Xu and Chang 2007).

Statistical analysis

Data were expressed as mean ± standard deviation of three independent experiments. The significance of difference at p < 0.01 was determined by one‐way ANOVA with Tukey’s post hoc test using IBM SPSS Statistics software.

Results and discussion

Interference of endogenous lipids on lipase activity

The rice bran oil (RBO) has been recognized as a healthier oil due to its fatty acid composition. RBO is obtained from rice bran (RB), a by-product of the rice milling process, and contains 15–20% of oil and antioxidants. RB lipases are highly active even at high temperatures and hydrolyze the stored lipids to FFA (Bhardwaj et al. 2001). Hence, stabilization or inactivation of bran lipases is an important process to utilize RBO. The existing thermal and chemical treatments are compromising the loss of phytonutrients. Alternatively, understanding the structural and functional characteristics of RB lipases will be a promising strategy for bran stabilization by enzyme inhibition. Hence, we made an effort to characterize the RB lipases, but the interference from endogenous lipids was misleading both enzyme activity and detection of possible lipases (Fig. 1a). The ultracentrifuged RB supernatant was used as an enzyme source to measure lipase activity using physiological substrates such as MAG, DAG, TAG and phospholipids. Unexpectedly, the presence of endogenous lipids in the RB protein was too high, and it interfered with enzyme activity. The FFA release was observed even in the negative control or “no-substrate control” along with other lipids. It revealed the presence of intrinsic substrates in the enzyme source (Fig. 1b, c) which is hydrolyzed by lipases and released FFAs. To confirm the presence of endogenous lipid substrate, the lipase assay was performed without the addition of the substrate by incubating equal concentration of RB lysate at different time points. Surprisingly, we observed the time-dependent FFA release, which confirms the utilization of intrinsic substrates by lipases (Fig. 1d). Figure 1e shows the amount of total fatty acids present in RB lysate. Therefore, delipidation of the protein samples is mandatory for the accurate prediction of lipase activity using physiological substrates. Although the organic solvents were extensively used as delipidation agents, it denatures the enzymes (Cham and Knowles 1976; Vaisar 2009). Agnese et al (1983), reported the clarification of lipids from serum samples using Freon 113, Dextran sulfate 500-S and Aerosi1 380. However, it has been limited to serum samples with an extended incubation period and the functional validation of the lipases is lacking.

Fig. 1.

Fig. 1

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Interference of endogenous lipids on lipase activity. a Schematic representation of lipid inference in enzyme assay. The intrinsic lipids compete with the substrate subsequently catalyzed by lipases to product and mislead the final activity. The lipase assay was performed at 37 °C for 30 min with 50-µM MAG (b) or DAG (c) in the presence of 50 µg of RB protein. The reaction was stopped followed by extraction of lipids, and the lipids were separated on a silica-TLC plate using petroleum ether: diethyl ether: acetic acid (70:30:1, v/v) as the solvent system. •• RB lysate was boiled for 5 min and used as an enzyme source. d RB lysate was incubated at different time intervals without substrate, and FFA release was quantified by a fluorometric assay. Statistically different (p < 0.01) against the 0 time point. e Quantification of total fatty acid in RB lysate. Values are mean ± SD from three independent experiments

Solvent-free ASG treatment significantly reduces the endogenous lipids content

The delipidation of the RB sample was performed for 30 min at 4 °C with different ratios of ASG ranging from 1:2 to 1:10 (w/v) and a minimum 1 mg/mL of protein was preferred to maintain in the measured volume. The result revealed that the ratio of ASG and protein were positively correlated with the amount of delipidation. The optimal delipidation was observed with 1:2 (w/v) of ASG and RB protein, respectively (Fig. 2a). Further, the Celite and Florisil have been reported for isolation of lipids from animal tissues (Marmer and Maxwell 1981; Toledo Netto et al. 2012). We also have tried to remove RB lipids in our experimental conditions using these two compounds. Although florisil was able to remove a minor fraction of lipids, celite was ineffective in lipid clarification in our experimental condition (Fig. 2b, lanes 3 and 4). Interestingly, ASG treatment significantly removed the endogenous lipids as compared with those two compounds (Fig. 2b). The quantification of total FFA content by GC/MS analysis of delipidated samples showed 85–90% efficacy as compared with untreated samples in our experimental condition (Fig. 2c). Further, it was confirmed by quantification of total FFA by fluorometric assay, and similar results were observed (Fig. 2c). The delipidation efficiency of the adsorbents such as silica, florisil and celite typically depends on the lipophilic groups present in their chemical structure. Further, surface area as well as the average particle size of the adsorbents is crucial for its delipidation capacity (Taspinar and Ozgul-Yucel 2008; Toledo Netto et al. 2012). The delipidation of serum samples was reported using the solvent-free methods (Onder et al. 2018; Sharma et al. 1990; Dimeski and Jones 2011; Castro et al. 2000). However, the efficiency of lipid removal was low particularly for TAG. The maximum reduction of TAG was only 21.2%; whereas, total cholesterol content and FFA was 54.4% and 30%, respectively (Barrera et al. 2018). Moreover, reported delipidation methods were concerned about the removal of cholesterol, TAG and FFA, and there was no evidence on the impact of delipidation on polar lipids such as phospholipids. The ASG treatment effectively removed the total phospholipids in the RB samples (Fig. 2d). Further, the delipidated sample was optically clear, free from flocculation and turbidity; whereas, the control sample was more dense and cloudy (Fig. 2f).

Fig. 2.

Fig. 2

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Delipidation of RB lysate. a Delipidation of RB lysate with different ratio of ASG. Equal concentration of protein was taken from control and treated sample followed by lipid extraction using chloroform: methanol (1:2, v/v). The extracted lipids were separated on a silica-TLC plate using petroleum ether: diethyl ether: acetic acid (70:30:1, v/v) as the solvent system. b Neutral lipid profile of delipidated RB lysate in comparison with florisil and celite. c Quantification of total fatty acids in delipidated RB lysate. Insert represents the NL profile of RB lysate. d Phospholipid profile of delipidated RB lysate. e Levels of total polyphenols in delipidated RB lysate. f The optical clarity of RB lysate after delipidation. Values are mean ± SD from three independent experiments. *Statistically different (p < 0.01) against the control

Delipidation of RB proteins retains enzyme activity

The important objective of delipidation was to maintain the protein functional, and it was validated by enzyme assay and ABPP. The lipase activity was monitored in the delipidated protein samples using various physiological substrates at 37 °C for 30 min. Lipids were extracted, separated on TLC which showed that there was no interference of endogenous substrates. Prominent lipase activity was observed with all substrates, and the product formation was correlated with the reduction of substrates (Fig. 3b–c). We performed the assay using phospholipids mainly phosphatidylcholine (PC) and lysophosphatidylcholine (LPC), since after TAG, PC is the predominant phospholipid in RB (Glushenkova et al. 1998). The phospholipase assay with PC substrate showed the consecutive hydrolysis of PC followed by LPC (Fig. 3d). It was further confirmed with enzyme assay using LPC as a substrate. It evidences that the ASG treatment retains the protein at a native state which confirms the functional integrity of the lipases.

Fig. 3.

Fig. 3

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Improved lipase activity and ABPP signal detection upon delipidation. a Schematic representation of enhanced lipase functions by delipidation. b TLC profile of MAG lipase assay using delipidated RB lysate. MAG lipase assay was performed with control and treated sample as a protein source for 30 min at 37 °C with 50-µM MAG. The reaction was stopped by extracting lipids, and the lipids were separated on a silica-TLC plate using petroleum ether: diethyl ether: acetic acid (70:30:1, v/v) as the solvent system. c Quantification of enzyme activity by measuring FFA release from MAG, DAG, and TAG. Insert represents the TLC profile of lipase assay using TAG substrate. Values are mean ± SD from three independent experiments. d TLC represents the phospholipase activity of delipidated RB lysate using PC and LPC as substrate. Phospholipase assay was performed with control and treated sample as a protein source for 30 min at 37 °C with 50-µM PC or LPC as a substrate. The reaction was stopped, and lipids were resolved on a TLC plate using chloroform: methanol: ammonia (65:25:5, v/v) as the solvent system. e ABPP profile of RB lysate. The fluorescent gel image represents the presence of possible hydrolases. After the in-gel fluorescent scanning, the gel was stained with CBB

Delipidation enhances the ABPP probe binding and improves the signal

The second target of our study was to detect the presence of possible RB lipases using ABPP probes. As mentioned earlier, the intrinsic lipids lead to the loss of the ABPP signal in a gel-based platform followed by a reduction of peptide count during LC/MS analysis (Galmozzi et al. 2014). However, there was no experimental evidence on the influence of endogenous lipids during ABPP labeling. In ABPP labeling, we observed the hindrance of the active site and blurred signals with control RB protein samples. Interestingly, the ASG-treated samples showed an improved ABPP signal with sharp protein bands (Fig. 3e). Besides that, we could detect prominent signals around 90-kDa and 35-kDa region in the delipidated samples. It suggests that in the control sample, the active sites were shared or occupied with endogenous lipids leading to the reduction of probe labeling. However, the ASG treatment might adsorb a sizable number of charged amino acids, especially arginine-containing proteins (Castro et al. 2000) and we also observed a minor amount of protein loss but that did not affect the ABPP labeling. In addition to lipids, bran contains a higher amount of phenolic compounds which contribute up to 95% of the total phenolic acids in the rice grain (Zhou et al. 2004).The reduction of protein labeling might be due to the interference of polyphenolic compounds along with lipids. The polyphenolic components were reported as potent lipase inhibitors (Buchholz and Melzig 2015). On silica treatment, there was a significant reduction of TPC content in ASG-treated samples as compared with control (Fig. 2e). It suggests that the enhanced ABPP labeling and improved optical clarity were due to the reduction of TPC along with the delipidation.

Delipidation of mouse adipose tissues

Further, the ASG mediated delipidation was validated in a lipid-rich animal sample such as mice adipose tissue (AT). Since AT stores ~ 80% of total body fat, isolation or purification of the proteins particularly lipases from adipose tissue in active state is still difficult (Tornqvist and Belfrage 1976). The solvent-based delipidation followed by detergent-aided solubilization leads to loss of functional integrity and is highly incompatible to mass spectrometry analysis (Sajic et al. 2011). The optimized delipidation condition for bran was followed for AT protein also and we observed the improved optical clarity upon delipidation (Fig. 4a). The ASG treatment significantly reduced the endogenous lipids and it was further confirmed by fatty acid quantifications (Fig. 4b). The functional integrity of the delipidated protein was validated by measuring the lipase activity using a physiological substrate, triacylglycerol (Fig. 4c). The ABPP labeling of delipidated AT samples showed the enhanced signal along with the detection of additional proteins (Fig. 4d). ABPP-based proteome approach has been well documented for the functional characterization of lipases and drug discovery (Galmozzi et al. 2014). However, its the first report on the enhancement of ABPP probe labeling in adipose tissue by solvent-free delipidation.

Fig. 4.

Fig. 4

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Delipidation of mice adipose sample. Mice adipocyte tissue was treated with ASG under the optimized conditions. a Appearance of control and delipidated AT samples. Total lipids were extracted from the control and treated samples, and the extracted lipids were separated on a silica-TLC plate using petroleum ether: diethyl ether: acetic acid (70:30:1, v/v) as the solvent system. b Quantification of FFA in delipidated AT lysate. Insert represents the NL profile. c TAG lipase activity of delipidated AT lysate. TAG lipase assay was performed with control and treated sample as a protein source for 30 min at 37 °C with 50-µM TAG. The reaction was stopped by extracting lipids, and the FFA release was quantified by a fluorometric assay. d ABPP profile of AT lysate. The fluorescent image represents the presence of possible hydrolases. Statistically different (p < 0.01) against the control. Values are mean ± SD from three independent experiments

Conclusion

We have demonstrated a simple and solvent-free delipidation procedure using ASG without affecting the functional integrity of the proteins. The advantages of the method are: easily adaptable protocol, efficiently removes the interfering endogenous lipid and polyphenolic compounds, and highly compatible with subsequent proteomic analysis such as enzyme assay and ABPP. Further, this approach might have a significant impact on the identification of novel lipases and the discovery of small-molecule inhibitors using the ABPP strategy.

Acknowledgements

We are grateful to Prof. Ram Rajasekharan for his constant support and encouragement. We thank Dr. A. Jayadeep, CSIR-CFTRI for providing the fresh Oryza sativa (IR64) seeds for the study. We appreciate Dr. Arun Kumar V, CSIR-CFTRI for his constructive criticism of the manuscript.

Author contributions

PV conceived the original research plans and supervised the experiments. AKD carried out the experiments. PV and AKD analyzed and discussed the data. PV and AKD wrote the manuscript.

Funding

Science and Engineering Research Board IFA14-LSPA28 and ECR/2015/000564.

Compliance with ethical standards

Conflict of interest

Authors have no conflict of interest to declare.

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