Complete Protein Protection from Proteases and Phosphatases

Protease, phosphatase and combination inhibitor tablets provide a convenient format to protect proteins from degradation and dephosphorylation

by Kay Opperman, Ph.D.; Hai-Yan Wu, Ph.D.; Barbara Kaboord, Ph.D.; Monica Noonan, M.S. - 04/18/12

Proteases and phosphatases serve many metabolic and regulatory functions; however, upon cell lysis, the once compartmentally contained enzymes are capable of mass protein degradation and dephosphorylation. The preservation of proteins from protease and phosphatase activities after cell or tissue lysis is essential for many research applications. The Thermo Scientific Pierce Protease, Phosphatase, and combination Protease and Phosphatase Inhibitor Tablets are broad-spectrum formulations ideal for preventing proteolytic degradation and dephosphorylation during cell lysis and protein extraction.

Proteases are a ubiquitous class of enzymes that hydrolyze protein peptide bonds. Proteases are divided into two broad categories based on cleavage site and then subdivided based on the active site and pH preferences. The exopeptidases (carboxypeptidases and aminopeptidases) cleave peptides proximal to the amino or carboxy termini of the substrate; endopeptidases (serine, aspartic, cysteine and metalloproteinases) cleave the peptide distal to the terminus. Plants contain the largest distribution of proteases (44%), followed by bacteria (18%), fungi (15%), animals (11%), algae (7%) and viruses (4%); however, the variety, distribution and compartmentalization of proteases vary among the different organisms.

Phosphatases are hydrolase enzymes that remove phosphates from proteins and other molecules. The balance of phosphorylation/dephosphorylation is vital to many cellular signaling pathways, including signal transduction, cell division and apoptosis. Phosphatases are categorized based on sequence, structure, and catalytic function. Protein phosphatases generally target serine, threonine and tyrosine phosphorylation during cell signaling. Acid phosphatases are localized in acidic lysosomes, and alkaline phosphatases are active in more basic cellular environments. The phosphatases are also ubiquitous, but depending on function, are preferentially concentrated in certain cell types and tissues.

We evaluated the effectiveness of the Pierce Protease, Phosphatase, and the combination Protease and Phosphatase Inhibitor Tablet formulations using a variety of protease and phosphatase substrates. We compared tablet performance to tablets from other suppliers. Our formulations protected proteins from protease and phosphatase degradation and performed favorably compared to other commercial preparations.

Protease inhibition

Protease assays are somewhat confounding in that general protease substrates are not a true assessment of the contribution of each individual inhibitor activity. Similarly, each formulation must be compared using the same conditions, such as the same extract source, preparation, and protein concentration. After evaluating many fluorescent substrates, we chose assay substrates for cysteine proteases (papain) and serine proteases (trypsin) and used mouse pancreatic extract (1mg/mL) as the positive control. Cleavage of the cysteine protease substrate results in fluorescence at 460nm, and cleavage of the serine protease results in fluorescence at 520nm.

We compared protease inhibition by the Pierce Tablet formulations (Table 1) to other commercial formulations. Our tablet formulation nearly completely inhibited papain activity (98% and 94% inhibition) in pancreatic extracts and performed better than other commercial formulations (Figure 1). Tablet formulations inhibited approx. 80% of serine protease activity. Similarly, the tablets inhibited > 90% of papain activity in different cell lines and tissues lysates (Figure 2).

A.

B.

Figure 2. The tablet formulation inhibits protease activity in different tissue lysates and cell lines. Protease activity of cell and tissue lysates (1mg/mL) was determined in the presence or absence of the prepared Pierce Protease Inhibitor Tablet. The percentage of protease inhibition is indicated.

Phosphatase inhibition

To evaluate effectiveness of the phosphatase inhibitor tablets, we performed western blot analysis on phosphorylated targets and assessed phosphatase activity using assays for acid, alkaline and protein phosphatase. We detected phosphorylated AKT and PDGF from NIH 3T3 cells that were stimulated with PDGF after serum-starvation (Figure 3A) and lysed in the presence of the prepared inhibitor tablets. Furthermore, we detected phosphorylated ERK1/2 in homogenized tissues protected with the inhibitor (Figure 3B). Our results indicate that cell lysis in the presence of the inhibitor can preserve phosphorylation.

To assess phosphatase activity, the tablets were reconstituted and incubated with mouse brain extract and fluorescent phosphatase substrates. Cleavage of the phosphate results in fluorescence at 528nm. Our phosphatase inhibitor tablets were able to inhibit 91% of the protein phosphatases, 71% of the acid phosphatases, and 95% of the alkaline phosphatases activities (Figure 3C).

Figure 3. Protein phosphorylation is preserved in cell and tissue extracts. Western blot analyses of relative levels of total and phosphorylated proteins from protein extracts prepared in the absence or presence of phosphatase inhibitors. Panel A: AKT and PDGFR in serum-starved, PDGF-stimulated (100ng/mL) NIH 3T3 cell extracts. Panel B: ERK1/2 in liver and spleen tissue extracts Panel C: The degree of inhibition for protein, acid and alkaline phosphatase activity was determined in mouse brain extract after treatment with Pierce Phosphatase Inhibitor Tablets or another commercially available phosphatase inhibitor tablet. Percent inhibition is indicated.

Combined protease and phosphatase inhibition

To evaluate the effectiveness of the Pierce Inhibitor Tablets that are formulated to inhibit both proteases and phosphatases, we measured protease and phosphatase activity in mouse brain extracts. The combination tablet formulations were able to inhibit most of the enzyme activity. The EDTA formulation was slightly more effective, inhibiting 87% and 96% of the protease activity and phosphatase activities, respectively (Figure 4). At the time of this study, the combination tablets had no commercially available comparisons.

Excellent protection against cellular proteases and phosphatases during protein sample preparation was achieved using the Pierce Inhibitor Tablets. The combined tablet is the only commercially available tablet formulation that provides protection against both proteases and phosphatases. The tablets are provided in vials and may be reconstituted just before use for maximum protection.

Cell Line and Tissue Lysis: HEK293, A549, and HepG2 cell lines were maintained according to ATCC guidelines. For cell lysis, cells were grown to 80-90% confluency, rinsed with ice-cold PBS and lysed with ice-cold Thermo Scientific M-PER Mammalian Protein Extraction Reagent (Part No. 78501), with or without inhibitors. The lysate was transferred to a tube, incubated on ice for ten minutes, and clarified by centrifugation for 10 minutes at 10,000 x g. Supernatants were stored at -80C until use. Frozen tissue (Pel-Freez Biologicals) was homogenized in Thermo Scientific T-PER Tissue Protein Extraction Reagent (Part No. 78510), with or without inhibitors, using a polytron. Lysates were clarified by centrifugation for 10 minutes at 10,000 x g. Supernatants were stored at -80°C until use.

Preparation of Pancreatic Extract: Pancreatic extract was prepared using 5-6 frozen mouse pancreases homogenized in borate buffer (pH 8.5) and clarified by centrifugation for 10 minutes at 15,000 x g. Supernatants were collected and protein concentration was determined using the Thermo Scientific Pierce 660nm Protein Assay Reagent (Part No.22662) and either used fresh or immediately frozen.

Cysteine Protease Inhibition: Inhibitor tablets were reconstituted according to the manufacturer’s instructions. Fluorescent papain substrate (100µM final) was coated onto black, 96-well microplates. Protease inhibitor (1X final) and pancreatic extract (diluted in borate buffer to 1mg/mL) was added to the plates and incubated for 1 hour at 37°C. Activity was measured using a Thermo Scientific VarioSkan Flash Plate Reader (excitation/emission: 380/460nm). The percent protease inhibition is indicated for each protease inhibitor formulation. Percent inhibition was calculated relative to the wells that contain substrate and extract without inhibitor tablet solution. Samples were assayed in at least triplicate.

Serine Protease Inhibition: Inhibitor tablets were reconstituted according to the manufacturer’s instructions. A mixture of protease inhibitor tablet solution (50µL, 3X), trypsin solution (50µL, 0.1 U/µL), and protease substrate solution (50µL) are added into a 96-well microplate. Reactions were incubated for 2 hours at 37°C and the fluorescence intensity was determined at Ex490/Em520nm using a Thermo Scientific VarioSkan Flash Plate Reader. The percent protease inhibition is indicated for each protease inhibitor formulation. Percent inhibition was calculated relative to the wells that contain substrate and trypsin solution without inhibitor tablet solution. Samples were assayed in at least triplicate.

Phosphatase Assays: Tissue extract was prepared using frozen mouse brain. Two brains were homogenized in Thermo Scientific T-PER Tissue Protein Extraction Reagent (Part No. 78510) and clarified by centrifugation for 10 minutes at 10,000 x g. Supernatants were collected and protein concentration was determined using the Pierce 660nm Protein Assay Reagent and either used fresh or immediately frozen. Inhibitor tablets were reconstituted in water. Fluorescent acid, alkaline or protein phosphatase substrate (MFP, FDP and FDP, respectively) was coated onto black 96-well microplates. Protease inhibitor (1X final) and brain extract (diluted in assay sample buffer to 0.4mg/mL) were added to the plates and incubated for 1 hour at 37°C. Activity was measured using a VarioSkan Flash Plate Reader (excitation/emission: 485/528nm). Percent inhibition was calculated relative to the wells that contained substrate and extract without inhibitor. Samples were assayed in at least triplicate.

Cell Culture and Growth Factor Stimulation: NIH 3T3 cells were grown to near confluency in DMEM/high-glucose media supplemented with sodium private, sodium bicarbonate, 10% fetal bovine serum and penicillin/streptomycin. The cells were washed with HBSS, and media was replaced with reduced serum media (1% FBS) and incubated for 48 hours at 37°C. Cells were stimulated with 100ng/mL of PDGF (Cell Signaling Technology, Inc.) for 10 minutes. Cells were lysed using M-PER Mammalian Protein Extraction Reagent (Part No. 78501) with or without phosphatase inhibitors. Lysates were clarified by centrifugation for 10 minutes at 15,000 x g. Supernatants were collected and protein concentration was determined using the Pierce 660nm Protein Assay Reagent.

Western Blotting: For each condition, 20µg of lysate was used to detect AKT, pAKT, PDGF, pPDGF, ERK1/2 and pERK1/2. After transfer onto nitrocellulose, membranes were blocked with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween*-20, and incubated overnight at 4°C with the primary antibodies. Signal was detected using the appropriate HRP-conjugated secondary antibodies, Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080) and exposure to film for 2 minutes.

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