Scalable production and biophysical characterization of an enzyme cocktail derived from human red blood cells

Abstract

Red blood cells (RBCs) play a critical role in oxygen and carbon dioxide transport, which is facilitated by RBC‐encapsulated hemoglobin (Hb) and carbonic anhydrase (CA). In addition, RBCs are constantly exposed to oxidative stress due to the intracellular reactive oxygen species (ROS) generated during Hb auto‐oxidation. Antioxidant enzymes within RBCs, such as superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (Prx), counteract ROS generation to protect the RBC from oxidative stress. Therefore, this study presents a scaled‐up method to extract an enzyme cocktail from lysed human RBCs, enriched with the major RBC enzymes with minimal Hb contamination. Using ethanol‐chloroform precipitation and multiple biophysical analyses (SDS‐PAGE, SEC‐HPLC, MALDI‐TOF, and LC‐MS/MS), the RBC enzymes were successfully separated from Hb in the hemolysate. The purified enzyme cocktail exhibited minimal Hb contamination and retained a significant amount of CA, and antioxidative enzymes like SOD and CAT. Therefore, this scalable RBC enzyme purification method provides an efficient approach for isolating RBC enzymes with broad biomedical relevance.

1. INTRODUCTION

Red blood cells (RBCs) play a crucial physiological role in transporting gaseous molecules, specifically oxygen (O₂) and carbon dioxide (CO₂), between the lungs and the tissues. The primary mechanism for gaseous ligand transport is the hemoglobin (Hb, a heme‐containing protein that constitutes approximately 90% of the dry weight of RBCs and accounts for 98% of their total protein content) molecule that is encapsulated within the RBC. ¹ , ² , ³ , ⁴ As RBCs are consistently exposed to elevated levels of O₂, they face the risk of generating reactive oxygen species (ROS). Within RBCs, Hb binds reversibly to O₂ while maintaining its heme iron in the ferrous state (Fe²⁺). However, Hb can undergo spontaneous auto‐oxidation, during which the heme bound O₂ molecule accepts an electron from the ferrous iron atom, resulting in the formation of the superoxide ion (O₂ ^•‐) and ferric Hb that is, methemoglobin (metHb). ⁵ , ⁶ Inside the RBC, metHb represents less than 1%–2% of the total Hb pool. This is tightly regulated by the nicotinamide adenine dinucleotide (NADH)‐dependent cytochrome b₅ reductase system, which continually converts oxidized Hb (HbFe³⁺) back to its functional ferrous form. ⁷ Under normal physiological conditions, the antioxidant enzyme system within RBCs effectively mitigates this oxidative threat by reducing metHb (HbFe³⁺) into Hb (HbFe²⁺) and dismutating O₂ ^•‐. Nonetheless, this dismutation process produces hydrogen peroxide (H₂O₂), which can react with another O₂ ^•‐ molecule, generating highly reactive hydroxyl radicals (OH^•) via the Fenton reaction. The presence of free heme and iron can exacerbate this process, leading to cellular protein and lipid damage. ⁸ In addition to the intrinsic vulnerability arising from Hb auto‐oxidation, the biochemical makeup of the erythrocyte membrane also heightens susceptibility to oxidative damage. ⁹ , ¹⁰ The membrane is rich in polyunsaturated fatty acids, which are highly sensitive to lipid peroxidation, and it contains membrane proteins with reactive thiol groups that are prone to oxidation. Thus, oxidation of the cell membrane can impair membrane stability and reduce cellular deformability.

RBCs are unique in that they lack nuclei and mitochondria, thus preventing them from synthesizing proteins. Despite this limitation, they have a lifespan of approximately 120 days in the circulation, thanks in large part to their robust antioxidant system. This system can be broadly categorized into enzymatic and non‐enzymatic components. The non‐enzymatic components include various small molecules such as ascorbic acid, α‐tocopherol, and reduced glutathione (GSH). α‐Tocopherol provides protection against peroxyl radicals, while GSH plays multiple roles, including maintaining Hb in its native Fe²⁺ oxidation state in RBCs and preserving RBC membrane thiols by neutralizing ROS and free radicals. ¹¹ , ¹² Beyond protecting themselves, erythrocytes also act as important systemic scavengers of ROS in the circulation. ¹³ They help neutralize ROS generated both endogenously and by other cell types, thereby supporting the redox balance in the bloodstream. This dual characteristic, serving as both a significant source of ROS through Hb auto‐oxidation and a crucial scavenger of ROS, underpins their central role as targets of oxidative stress. The interest in enzymology is not limited to erythrocyte antioxidant systems. Numerous studies have reported the purification and characterization of novel enzymes from diverse sources, including alkaline phosphatases, luciferases, proteases, and hydrolases, many of which show promise for industrial or biomedical use. ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²² , ²³ The enzymatic system of RBCs consists of non‐antioxidant as well as antioxidant enzymes. Carbonic anhydrase (CA) is the second most abundant protein inside the RBC and is a non‐antioxidant enzyme that catalyzes the reversible hydration of CO₂ to bicarbonate and hydrogen ions, which is essential for maintaining the acid–base balance in blood and tissues. ²⁴ , ²⁵ By regulating the pH, CA indirectly reduces oxidative stress, as pH deviations can negatively impact cellular function and metabolism. ²⁶ The RBC antioxidant enzyme system primarily consists of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and peroxiredoxin‐2 (Prx2). As previously mentioned, SOD catalyzes the dismutation of O₂ ^•‐ into H₂O₂, which is subsequently broken down into non‐toxic forms by three cytosolic enzymes: CAT, GPx1, and Prx‐2. CAT converts H₂O₂ into water and molecular oxygen, while GPx1 and Prx‐2 reduce H₂O₂ to water using a reducing equivalent of nicotinamide adenine dinucleotide phosphate (NADPH), regenerated by the glutathione/glutathione reductase and thioredoxin (Trx)/thioredoxin reductase (TrxR) systems. The activity of RBC antioxidant enzymes depends on both the concentration and source of H₂O₂. Prx2 and GPx1 efficiently remove low levels of H₂O₂ generated by Hb, while CAT, due to its higher K _m, becomes essential when peroxide concentrations increase. This tiered system allows RBCs to control oxidative stress under both basal and elevated conditions. ²⁷ Notably, the CAT enzymatic system is considered more energy‐efficient than GPx1 and Prx‐2 since it does not require an NADPH equivalent for H₂O₂ breakdown. ²⁸ , ²⁹ , ³⁰ , ³¹

Deficiencies or mutations in RBC antioxidant enzymes have been linked to various clinical abnormalities in humans and animals. ³² , ³³ , ³⁴ , ³⁵ , ³⁶ These pathologies often exhibit similar clinical profiles associated with Hb toxicity; for example, SOD‐deficient mice show elevated levels of metHb, lipid peroxidation, and hemolytic anemia. ³⁷ , ³⁸ Likewise, CAT‐deficient mice demonstrate heightened sensitivity to H₂O₂, leading to increased Hb oxidation and oxidative damage to RBCs. ³⁹ Prx2 deficiency has been associated with elevated Hb oxidation and hemolytic anemia, ³⁹ , ⁴⁰ while GPx deficiency can result in hemolysis and possibly hemoglobinuria due to lipid peroxidation. ⁴¹ Furthermore, altered or diminished antioxidant enzyme activity has been linked to various clinical consequences in aging populations and individuals with diabetes. ⁴² , ⁴³ Therefore, there are clinical indications that may benefit from supplementation of these antioxidant enzymes. In light of this projected need, this work describes a method to scale up purification of RBC enzymes with minimal Hb contamination.

The zinc‐induced Hb precipitation method uses zinc salts to selectively remove Hb from the hemolysate, leaving the remaining RBC enzymes in the supernatant. ⁴⁴ This approach removes ~97%–98% of Hb with RBC enzyme recoveries ranging from 40% to almost complete, though potential zinc‐induced alterations in enzyme structure warrant further validation. Previous reports showed that ethanol‐chloroform treatment of stroma‐free RBC hemolysate, followed by acetone precipitation, is able to isolate SOD but inactivates CAT. However, while acetone is effective in precipitating proteins, it does not adequately distinguish Hb from other RBC enzymes, which may cause increased Hb contamination in the RBC enzyme extract. Moreover, this method can reduce the yield of certain RBC enzymes. Typically, ammonium sulfate precipitation is required to isolate and maintain CAT activity. ⁴⁵ , ⁴⁶ However, an improved method for extracting SOD, CAT, and CA from the RBC hemolysate, while maintaining enzymatic activity, has been developed by incorporating a concentrated potassium phosphate buffer (3 M, pH 7.4) during the ethanol‐chloroform treatment step to promote the conformational stability of the extracted enzymes. ⁴⁷ , ⁴⁸ The ethanol‐chloroform method offers several advantages. ⁴⁸ It selectively removes Hb by driving it into the organic phase while retaining RBC enzymes such as SOD, CAT, and CA in the aqueous phase. This technique provides high enzyme recovery with minimal loss, is cost‐effective compared to commercial preparations, and remains simple to perform while being scalable for industrial applications. The method described in the literature used ~2 g of stroma‐free Hb (SFHb) as the starting basis for enzyme extraction. The method achieved 70%–100% activity retention for SOD and CA and over 100% for CAT under optimal conditions. While enzyme recovery was high, the mass of total enzymes and scale of extraction were limited. This corresponds to a small‐scale approach optimized for enzyme recovery and activity but not intended for large‐scale enzyme purification. ⁴⁸

Building on prior work, this current study aims to develop a scalable platform for the purification of an RBC enzyme cocktail consisting of SOD, CAT, Prx‐2, GPx‐1, and CA from RBC hemolysate for potential biomedical applications. The methodology incorporates advanced tangential flow filtration (TFF) for clarifying the RBC hemolysate, followed by ethanol–chloroform treatment to precipitate Hb while recovering RBC enzymes in the supernatant. The use of a 10 kDa hollow fiber filter facilitates purification and concentration of the RBC enzyme cocktail at a significantly larger scale compared to previous approaches in the literature. ⁴⁸ The current process begins with clarified RBC hemolysate (90 g/batch), which primarily consists of SFHb (~98% by mass) and trace amounts of RBC enzymes (~2% by mass). Across six large‐scale batches, an average enzyme cocktail recovery rate of 44.4% ± 8.2% was achieved, with yields of approximately 0.85 ± 0.16 g of RBC enzymes per batch. Although the RBC enzyme recovery rate is lower than that observed in small‐scale RBC enzyme purification methods, the absolute enzyme yield is substantially higher due to the significantly larger starting mass of RBC hemolysate used. In addition, the current study describes comprehensive biophysical analyses of the purified RBC enzyme cocktail to elucidate its composition and discusses potential therapeutic applications.

2. MATERIALS AND METHODS

Sodium phosphate monobasic (NaH₂PO₄), sodium phosphate dibasic (Na₂HPO₄), potassium phosphate monobasic (KH₂PO₄), potassium phosphate dibasic (K₂HPO₄), sodium chloride (NaCl), potassium chloride (KCl), and sodium hydroxide (NaOH) were obtained from Fisher Scientific (Pittsburgh, PA). Modified polyethersulfone (mPES) hollow fiber tangential flow filtration (TFF) modules with pore sizes of 10 kDa (S02‐S010‐05‐N), 50 kDa (N04‐S050‐05‐N), 500 kDa (N04‐S500‐05‐N), and 0.65 μm (N04‐E65U‐07‐N) were sourced from Repligen (Rancho Dominguez, CA). Individual RBC enzyme purifications were conducted using gravity columns from BioRad (Hercules, CA), with Q‐Sepharose Fast Flow resin for anion exchange chromatography and Octyl Sepharose 4 Fast Flow resin for hydrophobic interaction chromatography; both resins were obtained from Cytiva (Marlborough, MA). Expired human red blood cell (RBC) units were sourced from Impact Life (Davenport, IA). Amicon ultracentrifuge filters (10 kDa) were purchased from Millipore Sigma (St. Louis, MO).

2.1. RBC lysis and clarification of the RBC hemolysate

The human RBC washing and clarified RBC hemolysate (consisting primarily of human Hb (hHb) and trace amounts of RBC enzymes) purification processes were performed as described in the literature. ⁴⁹ In summary, 20 units of human RBCs were pooled and diluted to a hematocrit of 20% ± 2% using a 0.9 wt% saline solution. The mixture was stirred for at least 30 min to ensure uniformity. The RBC solution was washed over six diacycles using a 0.65 μm mPES hollow fiber filter with an additional 120 L of 0.9% saline solution. Following the wash step, the RBC solution was concentrated to 8 L, and RBC lysis was initiated by adding 10 L of phosphate buffer (3.75 mM, pH 7.4). The solution was stirred at 4°C for 1 h to achieve complete cell lysis. It was then passed through a 500 kDa hollow fiber filter to remove membrane fragments and large aggregates. The clarified RBC hemolysate, mostly consisting of Hb and trace amounts of RBC enzymes, was concentrated using a 50 kDa hollow fiber filter to over 200 mg/mL and stored at −80°C for future use.

2.2. RBC enzyme cocktail extraction

RBC enzyme cocktail extraction from the clarified RBC hemolysate was carried out as previously described in the literature. ⁴⁸ In brief, 90 g of freshly thawed clarified RBC hemolysate was mixed with 166.6 mL of potassium phosphate buffer (PPB) (3 M, pH 7.4), then diluted with phosphate‐buffered saline (PBS) (0.1 M, pH 7.4) to target a total protein concentration of 90 mg/mL. The mixture was stirred on ice for 5 min to ensure homogeneity. Subsequently, 333.3 mL of a cold ethanol‐chloroform solution (67.5:32.5) was slowly added to the clarified RBC hemolysate solution at a rate of 5 mL/min. After the addition was completed, the mixture was stirred for another 30 min and centrifuged to separate the hHb precipitate from the RBC enzyme‐containing supernatant. The first centrifugation step was performed at 10,000 rpm and 4°C for 20 min to yield a bright red hHb protein pellet. The RBC enzyme‐containing supernatant was then filtered through a 0.2 μm disk filter to remove any protein aggregates. Finally, the filtered RBC enzyme‐containing supernatant was mixed 1:1 with PBS (pH 7.4) and subjected to 10 diacycles over a 10 kDa hollow fiber filter with PBS (pH 7.4). The purified enzyme cocktail was concentrated to under 100 mL and stored at −80°C. The schematic of the large‐scale RBC enzyme extraction process is shown in Figure 1.

FIGURE 1.

Schematic representation of large‐scale RBC enzyme extraction process. The clarified RBC hemolysate was mixed with ethanol‐chloroform for selective Hb precipitation and centrifuged to separate the Hb pellet from the RBC enzyme‐rich supernatant. The supernatant was filtered using a 10 kDa hollow fiber TFF membrane, the retentate was concentrated, and stored at −80°C.

2.3. SDS‐PAGE analysis

Pre‐cast 4%–20% Novex tris‐glycine mini protein gels (Invitrogen, Waltham, MA) were used in conjunction with the Novex mini gel tank (Waltham, MA) for SDS‐PAGE analysis. Sodium dodecyl sulfate (SDS) running buffer (10×) was obtained from Invitrogen (Waltham, MA). Samples were diluted to 1 mg/mL of protein in phosphate‐buffered saline (PBS, 3.75 mM, pH 7.4) and mixed at a 1:1 ratio with SDS. The mixture was then heated at 85°C for 10 minutes. For analysis under reducing conditions, 4 μL of 1 M dithiothreitol (DTT) was added before heating. A protein ladder (5 μL) and samples (20 μL) were loaded onto the gel and run at 220 V for 40 min. The gels were stained with Coomassie blue (Thermo Fisher, Waltham, MA) for 40 min, followed by overnight destaining in a solution composed of 60% deionized water, 30% methanol, and 10% acetic acid. Densitometric analysis was conducted using the GelQuantNet software from biochemlabsolutions.com to assess the protein composition.

2.4. MALDI‐TOF analysis

MALDI TOF analysis of the precursor clarified RBC hemolysate, the RBC enzyme cocktail, and the protein pellet extraction was carried out as previously described in the literature. ⁵⁰ Briefly, a solution of α‐cyano‐4‐hydroxycinnamic acid (CHCA) was prepared by dissolving 10 mg of CHCA in 1 mL of 50% v/v acetonitrile containing 0.1% trifluoroacetic acid. Samples were diluted to a protein concentration of 1 mg/mL in 3.75 mM PBS, pH 7.4. The samples were then mixed with the CHCA matrix and 0.1 M HCl in a 1:5:1 ratio. Two microliters of the mixture were loaded onto a matrix‐assisted laser desorption/ionization (MALDI) plate and analyzed using a Bruker Microflex matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometer (Bruker, Billerica, MA). Data were processed with the Bruker Flex Analysis software.

2.5. Trypsin digest LC–MS/MS analysis

Trypsin digest LC‐MS/MS analysis of the precursor clarified RBC hemolysate, RBC enzyme cocktail, and protein pellet extraction was carried out as previously described in the literature. ⁴⁹ , ⁵⁰ Trypsin digest LC‐MS/MS analysis was performed at the Comprehensive Cancer Center's Mass Spectrometry and Proteomics facility at The Ohio State University. Enzyme samples were resuspended in a 50 mM ammonium bicarbonate solution and incubated at 65°C for 15 min with DTT. Afterward, the samples were incubated with iodoacetamide in the dark at room temperature. Sequencing‐grade modified trypsin (Promega, Madison, WI) was then added at a 1:50 ratio (trypsin: sample), and the mixture was incubated overnight at 37°C. The reaction was quenched with 50 μL of 0.1% formic acid (FA), and the samples were dried using a SpeedVac (Eppendorf, Enfield). Samples were resuspended in FA, and the final concentration was measured using a Nanodrop (Fisher, Pittsburgh, PA) before undergoing liquid chromatography–tandem mass spectrometry (LC‐MS/MS) analysis (Fisher, Pittsburgh, PA). Data analysis was conducted using the Scaffold 5 software (Proteome Software, Portland, OR).

2.6. Total protein concentration

The total protein concentration was determined using the Bradford assay. ⁵¹ The total RBC enzyme yield was calculated by dividing the total mass of purified RBC enzymes by the starting mass of RBC hemolysate and multiplying the ratio by 100.

2.7. SEC‐HPLC analysis

The molecular weight (MW) of the clarified RBC hemolysate consisting primarily of hHb and the RBC enzyme cocktail was estimated using size exclusion chromatography (SEC) on a high‐performance liquid chromatography (HPLC) system, as described in the literature. ⁵² , ⁵³ , ⁵⁴ The separation was carried out using an Ultimate 3000 system equipped with an Acclaim SEC‐1000 column (4.6 × 300 mm) from Thermo Fisher Scientific (Waltham, MA). The mobile phase consisted of PBS (3.75 mM, pH 7.4) and was delivered at a constant flow rate of 0.35 mL/min for all chromatographic runs. During each run, UV–visible absorbance spectra were recorded continuously across the spectral range 200–700 nm. For the clarified RBC hemolysate (primarily composed of hHb), the absorbance was monitored at 413 nm (Soret peak), while the absorbance was monitored at 280 nm for the purified RBC enzyme cocktail. The MW of Hb was estimated using the calibration curve supplied by the SEC column manufacturer, adjusted to account for system holdup times. This calibration was further verified in‐house with a protein ladder spanning 50–250 kDa. The apparent MW of hHb was calculated according to Equation 1:

$$\mathit{MW}\left(\mathit{Da}\right)={10}^{-0.7339t+t_c+t_g+t_1}$$ (1)

where t is the measured elution time, t _c represents the holdup time of the analytical column (11.2 min), t _g corresponds to the holdup time of the guard column (0.45 min), and t _l accounts for the holdup time in the lines (0.2 min).

2.8. Purification of catalase, superoxide dismutase, and carbonic anhydrase from the RBC enzyme cocktail using gravity column chromatography

The purified RBC enzyme cocktail served as the starting basis for the purification of CAT and SOD using anion exchange (5 mL Q Sepharose resin) gravity column chromatography. The column was first equilibrated with a binding buffer containing 25 mM Tris (pH 8.0) and 5 mM NaCl. The purified RBC enzyme cocktail was then loaded onto the column at a controlled flow rate to maximize protein binding to the resin. After the protein binding step, the column was washed with 5 column volumes (CVs) of the binding buffer to remove unbound proteins from the resin. SOD and CAT were eluted using binding buffer supplemented with NaCl. SOD (eluted with 75 mM NaCl) and CAT (eluted with 100 mM and 150 mM NaCl) fractions were pooled separately and concentrated using a 10 kDa Amicon ultrafiltration unit. The purified enzymes were stored at −20°C for future use. CA was purified from the purified RBC enzyme cocktail using hydrophobic interaction (5 mL Octyl Sepharose) gravity column chromatography. The binding buffer (20 mM PB with 1.5 M ammonium sulfate, pH 7.0) and elution buffer (25 mM PB, pH 7.0) were used to equilibrate the column. After loading the purified RBC enzyme cocktail onto the column, unbound proteins were removed with 5 CVs of binding buffer. CA was then eluted in steps (90%, 80%, 70%, and 60% binding buffer). Pooled pure CA fractions were buffer exchanged into 50 mM PB (pH 7.4) and concentrated with a 10 kDa Amicon filter before storage at −20°C.

2.9. Catalase activity assay

The enzyme activity of purified CAT was determined using a CAT assay kit procured from Millipore Sigma (St. Louis, MO) according to the manufacturer's protocol (catalog # CAT100).

2.10. Superoxide dismutase activity assay

The enzyme activity of purified SOD was determined using a SOD assay kit procured from Millipore Sigma (St. Louis, MO) according to the manufacturer's protocol (catalog # CS0009).

2.11. Carbonic anhydrase activity assay

The enzyme activity of purified CA was determined using a CA assay kit procured from Millipore Sigma (St. Louis, MO) according to the manufacturer's protocol (catalog # ab284550).

2.12. Statistical analysis

Data are shown as mean ± SD. Statistical significance (p < 0.05) was assessed using Student's t‐test, one‐way ANOVA, and Tukey's post hoc test (JMP Pro Version 16, SAS Institute Inc., Cary, NC).

3. RESULTS

3.1. RBC enzyme analysis

RBC lysis was carried out on 20 pooled human RBC units, followed by TFF purification of hHb. The purified hHb was then frozen at −80°C for future use. The clarified RBC hemolysate primarily consisted of hHb, resulting in a concentrated hHb solution (225.6 mg/mL). For each large‐scale RBC enzyme cocktail (LS‐EC) extraction, 90 g of clarified RBC hemolysate at 90 mg/mL was used as the basis, as summarized in Table 1. Ethanol‐chloroform treatment of the clarified RBC hemolysate precipitated hHb, enabling the recovery of RBC enzymes in the supernatant. Across six LS‐EC batches, the supernatant was effectively free of hHb and yielded a total of 0.85 ± 0.16 g of RBC enzymes per batch, with an RBC enzyme recovery rate of 44.4% ± 8.2% compared to the total initial mass of RBC enzymes in the clarified RBC hemolysate.

TABLE 1.

Summary of the RBC enzyme cocktail extraction process, concentration, and yield.

Batch ID	Starting mass of total protein in the RBC hemolysate (g)	Initial hHb content (%)	Initial mass of RBC enzymes (g)	Recovered volume (mL)	Recovered total protein conc. (mg/mL)	Total mass of recovered protein (g)	Recovered hHb content (%)	Total Mass of recovered RBC enzymes (g)	RBC enzyme yield (%)
LS‐EC‐01	90	98.2	1.62	85	10.92	0.928	3.9	0.89	46.4
LS‐EC‐02	90	98.2	1.62	95	9.16	0.870	2.4	0.85	44.2
LS‐EC‐03	90	98.2	1.62	95	9.83	0.934	1.4	0.92	48.0
LS‐EC‐04	90	98.2	1.62	90	7.50	0.675	1.3	0.67	34.7
LS‐EC‐05	90	98.2	1.62	95	7.34	0.697	1.0	0.69	36.0
LS‐EC‐06	90	98.2	1.62	95	11.55	1.097	0.5	1.09	56.9
Average	90	98.2	1.62	92.5	9.4	0.867	1.8	0.85	44.4
Standard Deviation	0	0	0	4.2	1.7	0.159	1.2	0.15	8.2

3.2. SDS‐PAGE analysis of RBC enzyme cocktail

The protein composition of the purified RBC enzyme cocktail was analyzed by SDS‐PAGE under both non‐reducing and reducing conditions (with DTT). The six‐precursor clarified RBC hemolysates showed the presence of a high‐intensity band at ~16 kDa, corresponding to the α‐ and β‐subunits of hHb (Figure 2a, non‐reducing conditions lanes 1–3 and reducing conditions lanes 5–6). Additional faint bands at ~30 kDa align with the MW of CA (monomer).

FIGURE 2.

SDS‐PAGE analysis: (a) Precursor clarified RBC hemolysate (consisting of primarily hHb and trace amounts of RBC enzymes), with lanes 1–3 under non‐reducing conditions without DTT, and lanes 5–7 under reducing conditions with DTT. (b) Batches of the purified RBC enzyme cocktail, with lanes 1–5 under non‐reducing conditions without DTT, and lanes 7–11 under reducing conditions with DTT. (c) Protein pellet, with lanes 1–5 under non‐reducing conditions without DTT, and lanes 7–11 under reducing conditions with DTT.

In the purified RBC enzyme cocktail fraction (Figure 2b, non‐reducing conditions lanes 1–5 and reducing conditions lanes 7–11), the primary proteins detected were CA, CAT, SOD, and Prx‐2. CAT showed a band at ~60 kDa, consistent with its monomeric subunits. Prx‐2 appeared at ~22 kDa (monomer) and ~ 44 kDa (dimer) under reducing conditions. SOD exhibited a band at ~17 kDa, matching the MW of its monomeric form. A faint hHb band (~16 kDa) indicated the presence of hHb α‐ and β‐subunits, but at much lower levels compared to the protein pellet fraction (Figure 2c).

The protein pellet fraction, obtained after ethanol‐chloroform treatment, predominantly contained hHb, indicated by a strong band at ~16 kDa, corresponding to the α‐ and β‐subunits of hHb (Figure 2c, non‐reducing conditions lanes 1–5 and reducing conditions lanes 7–11). Additional faint bands at ~30 kDa and ~ 44 kDa align with the MW of CA (monomer) and the dimeric form of Prx‐2, respectively.

Densitometric analysis of the SDS‐PAGE data, performed using GelQuantNet software, revealed the protein composition of the precursor clarified RBC hemolysate, enzyme cocktail fraction, and pellet fraction. The clarified RBC hemolysate primarily consisted of hHb at 93.83% ± 1.53%, with CA constituting 6.17% ± 1.53%. No detectable levels of CAT, SOD, or PRX were observed in this fraction. The enzyme cocktail showed a significant enrichment of CA (96.1% ± 2.16%), along with small proportions of CAT (1.93% ± 1.08%), SOD (0.73% ± 0.39%), Prx (0.27% ± 0.18%), and hHb at (0.20% ± 0.06%). The pellet fraction, which remained after enzyme extraction, retained a notable amount of hHb (77.02% ± 11.24%) and CA (21.86% ± 11.04%), while Prx (1.05% ± 0.57%) was also detected. No measurable levels of CAT or SOD were present in this fraction.

3.3. MALDI‐TOF analysis of purified RBC enzyme cocktail

MALDI‐TOF mass spectrometry (MS) analysis of the precursor clarified RBC hemolysate (Figure 3a) predominantly detected hHb subunits, with the α‐globin chain at 15130 m/z and the β‐globin chain at 15,873 m/z. A minor peak for CA was observed at 30,950 m/z. The purified RBC enzyme cocktail, shown in Figure 3b, confirmed the presence of several antioxidant enzymes. A prominent peak at 28,749, 30,202, and 31,716 m/z corresponds to the three different isoforms of the CA enzyme. Additional low‐intensity peaks were observed at 59,695 m/z, corresponding to the monomeric forms of the CAT enzyme. The presence of hHb in the purified RBC enzyme cocktail was confirmed by two distinct peaks at 15,173 and 15,876 m/z, corresponding to the α‐ and β‐subunits, respectively. MALDI‐TOF MS analysis of the pellet (Figure 3c) primarily revealed hHb, with peaks at 15,138 m/z (α‐globin) and 15,882 m/z (β‐globin). Low‐intensity peaks were also observed for CA at 30,650 m/z. MALDI‐TOF analysis of the purified CA enzyme reveals the existence of a high‐intensity peak at 28,749 m/z, which is ascribed to the MW of CA (Figure 3d), alongside a low‐intensity peak in the [M + H]²⁺ ionization state at 14,338 m/z. The mass values obtained are consistent with those reported in the literature. ⁵⁵ MALDI‐TOF analysis of the purified SOD enzyme (Figure 3e) displays distinct high‐intensity peaks at 15,690 m/z, which correspond to its monomeric form, and a low‐intensity peak at 33,747 m/z, indicative of its dimeric state. These masses align with those previously reported in the literature. ⁵⁶ , ⁵⁷ Furthermore, MALDI‐TOF analysis of the purified CAT enzyme (Figure 3f) shows a high‐intensity peak at 59,681 m/z, which corresponds to its monomeric form. These protein masses are consistent with values in the literature. ⁵⁸

FIGURE 3.

MALDI‐TOF analysis. (a) The precursor clarified RBC hemolysate shows predominant Hb α‐ and β‐subunits with a minor CA peak. (b) The RBC enzyme cocktail reveals peaks corresponding to CA isoforms, CAT monomers, and residual Hb α‐ and β‐subunits. (c) The protein pellet is enriched in Hb α‐ and β‐subunits with a minor CA peak. (d) The purified CA enzyme displays a strong [M + H]¹⁺ ion peak at 28,749 m/z and a secondary [M + H]²⁺ ion peak at 14,338 m/z. (e) The purified SOD enzyme shows a major monomeric peak at 15,690 m/z and a weaker dimeric peak at 33,747 m/z, and (f) The purified CAT enzyme is characterized by a dominant monomeric peak at 59,681 m/z.

3.4. Trypsin digest LC–MS/MS analysis of purified RBC enzyme cocktail

To identify the protein composition of the precursor clarified RBC hemolysate, purified RBC enzyme cocktail, and protein pellet, samples were analyzed by trypsin digest LC‐MS/MS (Figure 4). We use emPAI (Exponentially modified protein abundance index) to evaluate the relative abundance of the protein within the same sample as described previously in the literature. ⁵⁹ Mass spectrometry analysis of the clarified RBC hemolysate revealed that hHb α‐ and β‐globin chains comprised ~80%–85% of the total protein, with δ‐globin contributing ~10%–15% and γ‐globin ~3%–5%. Additionally, various RBC antioxidant enzymes were detected, including SOD, CAT, Prx‐1, 2, and 6, CA‐1, 2, and 3, GPx‐1 (glutathione peroxidase), BLVRB (biliverdin reductase B), CYB5R3 (NADH‐cytochrome b5 reductase 3), and Trx (thioredoxin), accounting for 0.5%–1.0% of the total protein in the clarified RBC hemolysate. In the purified RBC enzyme cocktail, CA enzymes were most abundant, with CA‐1 and CA‐2 comprising 55%–65% and 15%–25% of the total protein, respectively. SOD and CAT were the second most abundant RBC enzymes in the purified RBC enzyme cocktail, with relative quantities of 2%–3% of the total protein. Other antioxidant enzymes (Prx‐1, 2, 6, GPx‐1, BLVRB, CYB5R3, and Trx) were present at <1%–2% in the purified RBC enzyme cocktail. Residual hHb subunits (α, β, δ, and γ) in the purified RBC enzyme cocktail accounted for only 0.5–1.0% of the total protein. Mass spectrometry of the protein pellet sample showed that hHb was the primary component, with hHb α‐ and β‐globin chains making up 60%–65%, δ‐globin 30%–35%, and γ‐globin 0.5%–1.0% of all proteins. Minor quantities of CA (~1%) and other antioxidant enzymes (0.5%–1.0%) were also detected.

FIGURE 4.

Trypsin digest LC–MS/MS analysis of the purified RBC enzyme cocktail, protein pellet, and clarified RBC hemolysate. In the clarified hemolysate, hHb subunits (α‐, β‐, and δ‐globin) dominated the protein profile (~95%), with antioxidant enzymes including SOD, CAT, CA, Prx, GPx‐1, BLVRB, CYB5R3, and Trx detected at <1%. The purified RBC enzyme cocktail was enriched in CA isoforms (CA‐1: 55%–65%, CA‐2: 15%–25%), with SOD and CAT each contributing 2%–3%, minor antioxidant enzymes <2%, and residual hHb <1%. The protein pellet primarily consisted of Hb subunits (α‐ and β‐globin: 60%–65%, δ‐globin: 30%–35%), with only trace levels of CA and antioxidant enzymes. The inset shows the composition of the low‐abundance proteins within the precursor clarified RBC hemolysate, the purified RBC enzyme cocktail, and the protein pellet.

3.5. SEC‐HPLC analysis of the purified RBC enzyme cocktail

To estimate the MW of the purified RBC enzyme cocktail, samples were analyzed using SEC‐HPLC (Figure 5). The clarified RBC hemolysate exhibited an elution time of 9.2 min, corresponding to an estimated average MW of 64.5 kDa corresponding to hHb. Analysis of the six purified RBC enzyme cocktail batches revealed two distinct peaks, with elution times at 9.6 and 8.6 min. The peak at 9.6 min indicated a protein with an average MW of 32 kDa, consistent with the MW of CA. The second peak, observed at 8.6 min, corresponded to a protein with an estimated MW of ~200 kDa, aligning with the MW of CAT (tetramer).

FIGURE 5.

SEC‐HPLC chromatogram of the clarified RBC hemolysate and RBC enzyme cocktail. The clarified RBC hemolysate shows a major peak at 9.2 min, corresponding to Hb (~64.5 kDa). In contrast, the purified enzyme cocktail displays two distinct peaks: one at 9.6 min (average MW ~32 kDa, consistent with CA) and another at 8.6 min (average MW ~200 kDa, corresponding to CAT tetramers). Chromatograms were monitored at an absorbance of 280 nm (total protein) and 413 nm (Soret peak of hHb).

3.6. Analysis of purified catalase, superoxide dismutase, and carbonic anhydrase enzyme from the purified RBC enzyme cocktail

SOD and CAT were purified from the purified RBC enzyme cocktail via a single‐step anion exchange separation using a Q‐Sepharose column. 1 mL of the RBC enzyme cocktail was diluted 5‐fold with 25 mM Tris buffer (pH 8.0) and loaded onto the column. After binding, extensive washing was performed to remove unbound proteins. SOD and CAT were eluted in a stepwise fashion by increasing the NaCl concentration in steps (25, 50, 75, 100, and 150 mM). As shown in the SDS PAGE (Figure 6a,b), SOD eluted at 75 mM NaCl, while CAT eluted at 100 mM and 150 mM NaCl. CAT displays a band at approximately 60 kDa, consistent with the MW of its monomeric subunits. SOD showed a band at approximately 17 kDa, consistent with the MW of its monomeric subunits. The purified proteins were pooled, concentrated using 10 kDa Amicon ultrafiltration units, and stored at −20°C until further use. The CA purification profile in Figure 6c shows that purified CA eluted at 90%, 80%, and 70% of buffer A (25 mM PB, 1.5 M (NH₄)₂SO₄), displaying a single band at an apparent molecular weight of ~30 kDa. Pure proteins were pooled, concentrated using 10 kDa Amicon filters, and stored at −20°C for future use.

FIGURE 6.

SDS‐PAGE analysis of the individually purified RBC enzymes: (a) Protein marker in lane 1, purified RBC enzyme cocktail before single RBC enzyme purification in lane 2, flow through showing unbound protein in lane 3, wash of unbound and loosely bound protein in lane 4, 25 mM NaCl eluted fractions collected in lanes 5–7, 50 mM NaCl eluted fractions collected in lanes 8–10. (b) 75 mM NaCl eluted fractions collected in lanes 2–4, 100 mM NaCl eluted fractions collected in lanes 5–7, 150 mM NaCl eluted fractions collected in lanes 8–10. (c) Protein marker in lane 1, purified RBC enzyme cocktail before individual RBC enzyme purification in lane 2, flow through showing unbound protein in lane 3, wash of unbound and loosely bound protein in lane 4, 90% of buffer A eluted fractions collected in lanes 5–7, 80% of buffer A eluted fractions collected in lanes 8–10, and 70% of buffer A eluted fractions collected in lanes 11–12.

3.7. Catalase, superoxide dismutase, and carbonic anhydrase activity analysis

RBC enzyme activity was measured across three different samples: the precursor clarified RBC hemolysate, the RBC enzyme cocktail, and individually purified enzyme samples, as shown in Figure 7 and Table 2.

FIGURE 7.

Enzyme activity analysis of the precursor clarified RBC hemolysate, the RBC enzyme cocktail, and the individually purified enzymes.

TABLE 2.

RBC enzyme activity in the precursor clarified RBC hemolysate, the RBC enzyme cocktail, and individually purified enzyme samples.

	Precursor clarified RBC hemolysate (n = 3)	RBC enzyme cocktail (n = 3)	Purified CAT (n = 9)	Purified CA (n = 3)	Purified SOD (n = 3)
CAT Activity (U/g)	109 ± 11	441 ± 123	13,972 ± 3764	NA	NA
CA Activity (U/g)	21 ± 0.4	54 ± 21	NA	1116 ± 54	NA
SOD Activity (U/g)	33,288 ± 3890	1,167,185 ± 67,099	NA	NA	2,530,110 ± 590,706

The CAT‐purified sample exhibited the highest enzymatic activity at 13,972 ± 3764 U/g, which was significantly greater than that observed in both the precursor clarified RBC hemolysate (109 ± 11 U/g, p < 0.0001) and the RBC enzyme cocktail (441 ± 123 U/g, p = 0.0001) samples. Although the RBC enzyme cocktail sample showed a higher average CAT activity compared to the precursor clarified RBC hemolysate, this difference was not statistically significant (p = 0.9853). The purified CA exhibited the highest activity at 1116 ± 54 U/g, which was significantly higher than both the precursor clarified RBC hemolysate sample (21 ± 0.4 U/g, p < 0.0001) and the RBC enzyme cocktail fraction (54 ± 21 U/g, p < 0.0001). While the RBC enzyme cocktail fraction showed moderately higher enzyme activity compared to the precursor clarified RBC hemolysate, this difference was not statistically significant (p = 0.4958). The precursor clarified RBC hemolysate sample exhibited the lowest SOD activity (33,288 ± 3890 U/g). In contrast, the RBC enzyme cocktail fraction showed a markedly higher activity of 1,167,185 ± 67,099 U/g, which was statistically significant compared to the precursor clarified RBC hemolysate (p = 0.0159). The highest SOD activity was observed in the SOD purified sample, reaching 2,530,110 ± 590,706 U/g. This represented a highly significant increase not only compared to the precursor clarified RBC hemolysate (p = 0.0003) but also when compared to the RBC enzyme cocktail fraction (p = 0.0067). In summary, these results suggest that individual purification of the RBC enzymes substantially enhanced their enzymatic activity. These results demonstrate a progressive elevation in enzyme activity from the precursor clarified RBC hemolysate to the RBC enzyme cocktail, and further to the individually purified enzyme samples.

4. DISCUSSION

This study successfully demonstrates the large‐scale purification of an RBC enzyme cocktail primarily consisting of CA and enriched with antioxidative enzymes, such as SOD, CAT, and Prx‐2. The use of ethanol‐chloroform precipitation enabled effective separation of hHb from the target enzymes, as evidenced by SDS‐PAGE, SEC‐HPLC, MALDI‐TOF, and LC‐MS/MS analysis.

Across six large‐scale batches, RBC enzyme yields averaged ~44%, corresponding to ~0.85 g of RBC enzymes, the method provides substantially greater material output and shows good reproducibility, with yields ranging from 34% to 57%. Compared with earlier small‐scale approaches, ⁴⁸ which prioritized enzyme activity over yield, the present workflow emphasizes scalability. The resulting RBC enzyme mixture, enriched in CA, CAT, and SOD, offers sufficient quantities for downstream applications. While some efficiency is sacrificed relative to small‐scale methods, this trade‐off supports preparative use, and future optimization of precipitation and recovery steps may further improve both yield and consistency. Additional limitations include possible enzyme denaturation, non‐specific extraction, and process constraints such as TFF membrane fouling, which may affect scalability and cost.

SDS‐PAGE results under both reducing and non‐reducing conditions showed that the RBC enzyme cocktail contained only a low‐intensity band corresponding to the α/β hHb subunits, whereas the protein pellet fraction displayed a high‐intensity band corresponding to these subunits. This indicates that the ethanol‐chloroform treatment denatured hHb, leading to its precipitation and enrichment in the pellet fraction. The protein pellet also showed low‐intensity bands for CA and Prx‐2 dimer, suggesting that some RBC enzymes denatured along with the hHb during the extraction process. However, the purified RBC enzyme cocktail solution retained CA, SOD, and CAT bands, which were mostly absent in the protein pellet. This difference reflects the selective retention of these enzymes in the soluble fraction, highlighting the efficiency of the extraction process for isolating intact CA and antioxidant enzymes. SEC‐HPLC analysis confirmed that two key proteins in the enzyme cocktail consisted of various CA isoforms and CAT.

MALDI‐TOF mass spectrometry analysis provided a detailed protein composition profile across different fractions (clarified RBC hemolysate, RBC enzyme cocktail, and protein pellet), highlighting the distinct molecular signatures of hHb subunits, CA isoforms, and key antioxidant enzymes. The precursor RBC hemolysate predominantly displayed strong signals corresponding to Hb subunits, with a minor peak for CA. In the purified RBC enzyme cocktail, the presence of multiple CA isoforms was confirmed, along with low‐intensity peaks associated with CAT. Despite the overall low Hb content in this fraction, distinct peaks corresponding to Hb subunits were still detected, likely due to the strong ionization efficiency of Hb in MALDI‐TOF analysis. The pellet fraction primarily exhibited Hb peaks, with some signal for CA. Analysis of the purified CA enzyme showed high‐intensity band corresponding to its molecular weight, and antioxidant enzymes revealed distinct monomeric and dimeric forms for SOD, while CAT was observed in its monomeric form. The strong ionization and high‐intensity signals of Hb, even in fractions with minimal contamination, highlight the sensitivity of MALDI‐TOF MS in detecting trace amounts of highly ionizable proteins.

Tryptic digest LC–MS/MS analysis confirmed the differential protein composition across the clarified RBC hemolysate, purified RBC enzyme cocktail, and protein pellet fractions. In both the clarified RBC hemolysate and the protein pellet, hHb subunits (α, β, δ, and γ) accounted for more than 95% of the total proteins, with only trace amounts of antioxidant enzymes present. In contrast, the RBC enzyme cocktail contained minimal hHb contamination, indicating successful removal of hHb during the purification process. The purified RBC enzyme cocktail was enriched with enzymes, with CA isoforms being the most abundant, followed by antioxidative SOD and CAT enzymes. The high abundance of CA suggests that it remained highly stable and soluble during the extraction process. Additionally, other antioxidant enzymes such as Prx‐1, 2, and 6, GPx‐1, BLVRB, CYB5R3, and thioredoxin (Trx) were present at low levels, contributing to the RBC protein cocktail's overall antioxidative potential.

The comparison of results from tryptic digest LC–MS/MS analysis and densitometric SDS‐PAGE analysis highlights differences in protein composition quantification across samples, particularly in the distribution of Hb and RBC enzymes CA, CAT, SOD, and Prx. Tryptic digest LC‐MS/MS analysis provided a highly sensitive and detailed proteomic profile, confirming that Hb subunits (α, β, δ, and γ) dominated both the clarified RBC hemolysate and protein pellet fractions, accounting for over 95% of total proteins. This technique also identified antioxidant enzymes in trace amounts, with CA being the most abundant enzyme in the purified RBC enzyme cocktail, followed by CAT and SOD, and lower levels of Prx isoforms and other redox‐active proteins. In contrast, SDS‐PAGE analysis, which relies on protein separation by MW followed by densitometric quantification, yielded slightly different proportions. It quantified Hb in the clarified RBC hemolysate at 93.83% ± 1.53%, with CA constituting 6.17% ± 1.53%, while CAT, SOD, and Prx were undetectable. In the enzyme cocktail, SDS‐PAGE detected CA at a higher proportion (96.1% ± 2.16%) and reported lower values for CAT (1.93% ± 1.08%), SOD (0.73% ± 0.39%), and Prx (0.27% ± 0.18%). These discrepancies likely arise due to the differences in detection sensitivity and specificity between the two techniques. LC‐MS/MS provides precise peptide‐level identification, allowing detection of low‐abundance proteins such as Prx, GPx, and Trx, whereas SDS‐PAGE is limited by its resolution and dynamic range, potentially underestimating minor components. The higher CA levels reported in the enzyme cocktail by SDS‐PAGE suggest potential overestimation due to staining intensity differences among proteins. Overall, the combination of both techniques provides complementary insights, with LC‐MS/MS offering a detailed proteomic composition and SDS‐PAGE providing a quantitative overview of major protein fractions.

The recovery of CAT, CA, and SOD enzymatic activity demonstrated a stepwise enrichment across processing stages. The precursor clarified RBC hemolysate contained the lowest enzymatic activity, reflecting the diluted starting material. Subsequent processing into the RBC enzyme cocktail yielded a modest to substantial increase in enzymatic activity depending on the enzyme, while chromatographic purification of individual RBC enzymes resulted in the greatest recovery of enzymatic activity. For CAT, the enzymatic activity increased approximately 4‐fold in the RBC enzyme cocktail compared to the clarified RBC hemolysate precursor but rose nearly 130‐fold upon chromatographic purification of CAT, underscoring that the majority of functional recovery occurred at the chromatographic purification stage of the enzyme. Similarly, CA activity showed only a ~ 2.5‐fold increase in enzymatic activity in the RBC enzyme cocktail, whereas chromatographic purification of CA yielded an over 50‐fold recovery relative to the precursor clarified RBC hemolysate, again demonstrating that substantial recovery in enzymatic activity was only achieved after chromatographic purification. Chromatographic purification of SOD further boosted enzymatic activity to ~76‐fold over the precursor clarified RBC hemolysate levels. This indicates that SOD activity was efficiently recovered even at the intermediate RBC enzyme cocktail stage, unlike CAT and CA, which required chromatographic purification for realization of maximal enzymatic activity. Overall, these results demonstrate that chromatographic purification of individual RBC enzymes is the critical determinant of enzyme recovery for CAT and CA, whereas SOD exhibited substantial recovery already at the RBC enzyme cocktail stage. The fold increases in enzymatic activity observed highlight the efficiency of the individual RBC enzyme chromatographic purification strategy in producing high‐activity RBC‐derived enzymes and suggest potential utility in applications where stable, enriched enzyme preparations are required.

The removal of hHb and enrichment of CA and antioxidant enzymes in the RBC enzyme cocktail demonstrate the effectiveness of the ethanol‐chloroform‐based extraction process. Minimal hHb contamination ensures that the RBC enzyme protein cocktail is suitable for applications where Hb interference is undesirable. The RBC enzyme cocktail holds significant promise for biomedical applications, particularly in treating pH and oxidative stress disorders. A notable example is the design of hemoglobin‐based oxygen carriers (HBOCs), including polymerized systems such as PolyHb‐CAT‐SOD and PolyHb‐CAT‐SOD‐CA. These multifunctional carriers are engineered to regulate both carbon dioxide/pH homeostasis and reactive oxygen species (ROS) simultaneously. ⁶⁰ , ⁶¹ In these formulations, the inclusion of CA enhances the rapid conversion between CO₂ and bicarbonate, thereby supporting acid–base regulation, while CAT and SOD act in tandem to neutralize oxidative damage. Such combined activity has been proposed as a therapeutic approach in hemorrhagic shock and ischemia–reperfusion injury applications, where disturbances in pH and bursts of ROS typically occur together. The RBC enzyme cocktail developed here could serve as a foundation for designing nanozymes nanoparticles that mimic the activities of SOD, CAT, and peroxidases. ⁶² Through their sequential enzymatic action, first converting superoxide radicals to hydrogen peroxide and then breaking it down into water and oxygen, these enzymes protect cells from oxidative stress. ⁶³ , ⁶⁴ , ⁶⁵ Such enzyme‐mimicking nanozymes offer promising therapeutic potential for diseases associated with elevated ROS levels. Previous studies have shown that an antioxidant mixture of vitamins A, D, E, and melatonin delivered via a thermosensitive hydrogel can effectively promote skin wound healing. ⁶⁶ Similarly, the RBC enzyme cocktail could serve as a promising basis for developing antioxidant‐loaded hydrogels aimed at enhancing topical wound healing, particularly for burn injuries.

5. CONCLUSION

In summary, this study provides a reproducible and scalable method for isolating a high‐quality RBC enzyme cocktail enriched with CA and key antioxidative enzymes, such as SOD, CAT, and Prx‐2, while achieving substantial removal of hHb. The ethanol‐chloroform precipitation method demonstrated strong selectivity for enrichment of non‐hHb enzymes, with an average yield of ~44% (~0.85 g of RBC enzymes per batch). Comprehensive analysis through SDS‐PAGE, SEC‐HPLC, MALDI‐TOF, and LC‐MS/MS, confirmed that the purified RBC enzyme cocktail is highly enriched in CA (70%–75%) and contains measurable levels of antioxidative enzymes such as CAT, SOD, and Prx isoforms in the soluble fraction with minimal hHb contamination. Enzymatic assays showed that SOD was efficiently enriched at the RBC enzyme cocktail stage (~35‐fold, which further increased ~76‐fold after chromatographic purification), whereas CA and CAT required chromatographic purification for substantial enzymatic recovery (~50‐fold and ~ 130‐fold, respectively), underscoring chromatographic purification as the key step for their functional enrichment. The major contribution of this work lies in demonstrating a large‐scale, preparative workflow for RBC enzyme isolation that balances yield, purity, and reproducibility, in contrast to earlier small‐scale methods optimized mainly for protein activity. Within the scope of this study, the findings highlight the potential of the purified RBC enzyme cocktail as a versatile biochemical resource in biomedical applications requiring pH control and antioxidant function without hHb contamination. The selective removal of hHb and retention of enzymes make this RBC enzyme protein cocktail a promising therapeutic tool for addressing oxidative stress‐related disorders.

AUTHOR CONTRIBUTIONS

All authors were involved in the conceptualization, experimental design, data curation, and formal analysis of the study. Mohd A. Khan, Griffin J. Beyer, Naomi Goosby, and Lilly Ortiz contributed to the purification and characterization of the materials. The initial draft of the manuscript was prepared by Mohd A. Khan and Andre F. Palmer. Additionally, Mohd A. Khan, Griffin J. Beyer, and Andre F. Palmer were responsible for manuscript revision, editing, and finalization for publication. Funding acquisition was managed by Andre F. Palmer.

FUNDING INFORMATION

This work was supported by National Institutes of Health grants R01HL156526, R01HL158076, R01HL159862, and R01HL162120 to A.F.P.

CONFLICT OF INTEREST STATEMENT

A U.S. provisional patent application was filed based on this work.

Khan MA, Beyer GJ, Goosby N, Ortiz L, Palmer AF. Scalable production and biophysical characterization of an enzyme cocktail derived from human red blood cells. Biotechnol. Prog. 2026;42(1):e70072. doi: 10.1002/btpr.70072

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.