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Enhancement of potency and stability of human extracellular superoxide dismutase.

Kim S, Kim HY, Kim JH, Choi JH, Ham WK, Jeon YJ, Kang H, Kim TY - BMB Rep (2015)

Bottom Line: We found that a significant fraction of overexpressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation.The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification.Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul 137-040; New Drug Development Center, Daegu-Gyungpook Medical Innovation Foundation, Daegu 701-310, Korea.

ABSTRACT
Cells express several antioxidant enzymes to scavenge reactive oxygen species (ROS) responsible for oxidative damages and various human diseases. Therefore, antioxidant enzymes are considered biomedicine candidates. Among them, extracellular superoxide dismutase (SOD3) had showed prominent efficacy against asthma and inflammation. Despite its advantages as a biomedicine, the difficulty in obtaining large quantity of active recombinant human SOD3 (rhSOD3) has limited its clinical applications. We found that a significant fraction of overexpressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation. Also, purified rhSOD3 was unstable and lost its activity very quickly. Here, we suggest an ideal preparative method to express, purify, and store highly active rhSOD3. The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification. Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage.

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Stabilization of rhSOD3. (A) Purified rhSOD3 lost its catalytic activity over time. The catalytic activity of rhSOD3 stored in PBS at different temperatures was monitored for 13 days. (B) The catalytic activity of rhSOD3 stored in PBS, 0.1% BSA, or 10% FBS at 37℃ is shown. (C) After 13 days of incubation, the amount of rhSOD3 was analyzed by Western blotting. (D) Freshly purified rhSOD3 were dialyzed under three different conditions; dialysis into PBS buffer, dialysis into PBS buffer after mixing 0.1% BSA with purified rhSOD3, and dialysis into PBS buffer containing 0.1% PEG. (E) Purified rhSOD3 was stored in PBS, 0.1% BSA, 1% BSA, 10% glycerol, or a protein stabilizing cocktail, and their catalytic activities were measured after repeated freeze/thaw cycles.
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Figure 003: Stabilization of rhSOD3. (A) Purified rhSOD3 lost its catalytic activity over time. The catalytic activity of rhSOD3 stored in PBS at different temperatures was monitored for 13 days. (B) The catalytic activity of rhSOD3 stored in PBS, 0.1% BSA, or 10% FBS at 37℃ is shown. (C) After 13 days of incubation, the amount of rhSOD3 was analyzed by Western blotting. (D) Freshly purified rhSOD3 were dialyzed under three different conditions; dialysis into PBS buffer, dialysis into PBS buffer after mixing 0.1% BSA with purified rhSOD3, and dialysis into PBS buffer containing 0.1% PEG. (E) Purified rhSOD3 was stored in PBS, 0.1% BSA, 1% BSA, 10% glycerol, or a protein stabilizing cocktail, and their catalytic activities were measured after repeated freeze/thaw cycles.

Mentions: We noticed that rhSOD3 was very unstable even after optimal purification procedures. Purified rhSOD3 lost almost half of its initial activity in PBS buffer at 37℃ within 7 days (Fig. 3A). Activity loss was also detected at low temperatures. Therefore, it was necessary to optimize the preparation and storage conditions to stabilize active rhSOD3. First, purified rhSOD3 was supplemented with 0.1% BSA or 10% FBS. Both 0.1% BSA and 10% FBS maintained the catalytic activity of rhSOD3 for up to 13 days at 37℃ (Fig. 3B), suggesting that BSA is sufficient to stabilize rhSOD3. Previous studies showed that Cu/Zn SOD can be fragmented, following copper release and/or non-enzymatic glycation (27, 28). Purified rhSOD3 disappeared almost completely after 13 days in PBS buffer (Fig. 3C), but 0.1 % BSA prevented this disappearance. This result suggested that rhSOD3 loses its activity due to fragmentation or degradation.


Enhancement of potency and stability of human extracellular superoxide dismutase.

Kim S, Kim HY, Kim JH, Choi JH, Ham WK, Jeon YJ, Kang H, Kim TY - BMB Rep (2015)

Stabilization of rhSOD3. (A) Purified rhSOD3 lost its catalytic activity over time. The catalytic activity of rhSOD3 stored in PBS at different temperatures was monitored for 13 days. (B) The catalytic activity of rhSOD3 stored in PBS, 0.1% BSA, or 10% FBS at 37℃ is shown. (C) After 13 days of incubation, the amount of rhSOD3 was analyzed by Western blotting. (D) Freshly purified rhSOD3 were dialyzed under three different conditions; dialysis into PBS buffer, dialysis into PBS buffer after mixing 0.1% BSA with purified rhSOD3, and dialysis into PBS buffer containing 0.1% PEG. (E) Purified rhSOD3 was stored in PBS, 0.1% BSA, 1% BSA, 10% glycerol, or a protein stabilizing cocktail, and their catalytic activities were measured after repeated freeze/thaw cycles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4352618&req=5

Figure 003: Stabilization of rhSOD3. (A) Purified rhSOD3 lost its catalytic activity over time. The catalytic activity of rhSOD3 stored in PBS at different temperatures was monitored for 13 days. (B) The catalytic activity of rhSOD3 stored in PBS, 0.1% BSA, or 10% FBS at 37℃ is shown. (C) After 13 days of incubation, the amount of rhSOD3 was analyzed by Western blotting. (D) Freshly purified rhSOD3 were dialyzed under three different conditions; dialysis into PBS buffer, dialysis into PBS buffer after mixing 0.1% BSA with purified rhSOD3, and dialysis into PBS buffer containing 0.1% PEG. (E) Purified rhSOD3 was stored in PBS, 0.1% BSA, 1% BSA, 10% glycerol, or a protein stabilizing cocktail, and their catalytic activities were measured after repeated freeze/thaw cycles.
Mentions: We noticed that rhSOD3 was very unstable even after optimal purification procedures. Purified rhSOD3 lost almost half of its initial activity in PBS buffer at 37℃ within 7 days (Fig. 3A). Activity loss was also detected at low temperatures. Therefore, it was necessary to optimize the preparation and storage conditions to stabilize active rhSOD3. First, purified rhSOD3 was supplemented with 0.1% BSA or 10% FBS. Both 0.1% BSA and 10% FBS maintained the catalytic activity of rhSOD3 for up to 13 days at 37℃ (Fig. 3B), suggesting that BSA is sufficient to stabilize rhSOD3. Previous studies showed that Cu/Zn SOD can be fragmented, following copper release and/or non-enzymatic glycation (27, 28). Purified rhSOD3 disappeared almost completely after 13 days in PBS buffer (Fig. 3C), but 0.1 % BSA prevented this disappearance. This result suggested that rhSOD3 loses its activity due to fragmentation or degradation.

Bottom Line: We found that a significant fraction of overexpressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation.The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification.Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul 137-040; New Drug Development Center, Daegu-Gyungpook Medical Innovation Foundation, Daegu 701-310, Korea.

ABSTRACT
Cells express several antioxidant enzymes to scavenge reactive oxygen species (ROS) responsible for oxidative damages and various human diseases. Therefore, antioxidant enzymes are considered biomedicine candidates. Among them, extracellular superoxide dismutase (SOD3) had showed prominent efficacy against asthma and inflammation. Despite its advantages as a biomedicine, the difficulty in obtaining large quantity of active recombinant human SOD3 (rhSOD3) has limited its clinical applications. We found that a significant fraction of overexpressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation. Also, purified rhSOD3 was unstable and lost its activity very quickly. Here, we suggest an ideal preparative method to express, purify, and store highly active rhSOD3. The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification. Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage.

Show MeSH
Related in: MedlinePlus