In the present study, the author have researched honey bees as a representative environmental indicator species.
bee poison(venom) is usefully used for anti-cancer, antibiotic, anti-inflammation, etc., and its main pharmacological component is melittin .
Melittin consists of 26 amino acids ...
In the present study, the author have researched honey bees as a representative environmental indicator species.
bee poison(venom) is usefully used for anti-cancer, antibiotic, anti-inflammation, etc., and its main pharmacological component is melittin .
Melittin consists of 26 amino acids and has a molecular weight of 2,846.46 Da. It ha a α-helx single strand for primary structure..
It is a amphiphillic membrane binding protein with amino acid sequence of NH2-GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2.
Melittin protein has the toxicity that disintegrates and necrotizes cell membrane composed of phospholipid at high concentration. The toxicity LD50 value of 50% mortality of a person's melittin is 2.8 mg/kg.
In order to use the bee venom melittin for medical purposes, this toxicity requires very deep attention.
In this study, we found that the partial hydrolysis of the bee venom melittin with the specific enzyme reduced the toxicity of melittin and maintained the abovementioned medical efficacy, and studied the cytotoxicity and molecular weight distribution of the bee venom melittin enzyme digestion peptides .
Melittin purified were obtained from bee venom according to the membrane filtration method, and digested with alcalase (EC NO. 232-752-2, A product Novozyme Corp.) At 37 ° C for 10 minutes, heat-treated to inactivate the enzyme, added cold ethanol(-20℃) to precipitate unreacted proteins, centrifuged to obtain supernatant, Enzymatic digestion peptide complexes were obtained by enriching and removing ethanol.
The safety of the obtained bee venom melttin enzyme digested peptide complex was evaluated by the protein stabilization test, the cell viability test and the degranulation inhibition test, and the bioactivity was evaluated by the anti - inflammatory test.
In addition, HPLC-MS was used to determine the molecular weight of the bee venom melittin enzyme-degrading peptide complex.
As a result, it was found that the protein denaturation can be inhibited even at a low concentration of 40 μg/ml of 50% (IC50) inhibition rate of the peptide complex obtained in this study, and in the cell viability test by MTT assay, no toxicity was observed below 200㎍/㎖ of peptides complex.
In addition, inhibition of β-hexosaminidase secretion also showed that the inhibitory effect on the secretion of β-hexosaminidase induced by a A23187 increased with dose.
In the anti-inflammatory test, cytokine TNF-α derived from TH1 cell and cytokine IL-4 derived from TH2 cell were measured, and the production of inflammatory cytokine was decreased according to the administration concentration.
In ionic curent chromatograms of HPLC-MS analysis, main peaks were observed at 2.4, 2.9, 3.6, and 4.6 minutes.
So, The authors decided to perform molecular weight measurements from 2.0 to 4.7 minutes.
As a result, the following results were obtained.
1). HPLC-MS analysis of the melitin enzyme digesting peptide complex prepared by this study was performed. As a result, there are 4 kinds of free amino acids, 7 kinds of di-peptides, 7 kinds of tri-peptides, 12 kinds of tetra-peptides, 12 kinds of penta-peptides, 9 kinds of hexa-peptides and hepta peptide, 3 kinds of nona-peptide, 3 kinds of deca-peptide, 1 type of hendeca-peptide, 1 kind of dideca-peptide, 3 kinds of trideca-peptide, 4 kinds of tetradeca-peptide, 7 kinds of pentadeca-peptide, 1 kind of hexadeca-peptide, respectively, and the molecular weight of each peptide was specified.
2). The major peaks of the melitin enzyme digestion peptide complexes prepared by this study were 2.4, 2.9, 3.6, and 4.6 minutes, respectively. The main compound molecular weights were 1482, 1556, 1383, 1038, 620, 1569, 607, 973, and high degree oligomers composed with at least 5 or more peptide bonds.
3). Compared to the type of peptide identified by G. Kreil (two heptapeptides, four tetra peptides, one pentapeptide, six types of tripeptide, and three dipeptides), the type of peptide I could see a lot.
In addition, it has been shown that the peptide complexes of melittin, trypsin and chymotrypsin hydrolyzates of G. kreil are composed of low molecular weight peptides, whereas the peptide complexes obtained in this study are composed of oligo peptides with molecular weight of 5 or more amino acids .
4). On the other hand, the enzymatic digestion of G. kreil showed a low molecular peptide complexes of 3-4 peptides on average, whereas the bee melittin enzyme digesting peptide complex in this study showed more than 5 peptides and more than 10 high oligo peptides. By this structure, it is thought that the peptide complex of this study is preserved without losing its physiological activity.
5). The peptides produced by this study inhibited the heat denaturation of albumin protein and inhibited the production of TNF-α and IL-4, which are inflammatory cytokines, without inducing cytotoxicity as a result of MTT assay.
From these results, it was possible to characterize the constituents and molecular weight of the active substance at the molecular level by applying HPLC-MS to a small amount of physiologically active substance which is small in quantity and difficult to be detected by conventional HPLC analysis. The results are expected to be useful as a means of evaluating small amounts of environmental pollutants in the natural world.
In the present study, the author have researched honey bees as a representative environmental indicator species.
bee poison(venom) is usefully used for anti-cancer, antibiotic, anti-inflammation, etc., and its main pharmacological component is melittin .
Melittin consists of 26 amino acids and has a molecular weight of 2,846.46 Da. It ha a α-helx single strand for primary structure..
It is a amphiphillic membrane binding protein with amino acid sequence of NH2-GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2.
Melittin protein has the toxicity that disintegrates and necrotizes cell membrane composed of phospholipid at high concentration. The toxicity LD50 value of 50% mortality of a person's melittin is 2.8 mg/kg.
In order to use the bee venom melittin for medical purposes, this toxicity requires very deep attention.
In this study, we found that the partial hydrolysis of the bee venom melittin with the specific enzyme reduced the toxicity of melittin and maintained the abovementioned medical efficacy, and studied the cytotoxicity and molecular weight distribution of the bee venom melittin enzyme digestion peptides .
Melittin purified were obtained from bee venom according to the membrane filtration method, and digested with alcalase (EC NO. 232-752-2, A product Novozyme Corp.) At 37 ° C for 10 minutes, heat-treated to inactivate the enzyme, added cold ethanol(-20℃) to precipitate unreacted proteins, centrifuged to obtain supernatant, Enzymatic digestion peptide complexes were obtained by enriching and removing ethanol.
The safety of the obtained bee venom melttin enzyme digested peptide complex was evaluated by the protein stabilization test, the cell viability test and the degranulation inhibition test, and the bioactivity was evaluated by the anti - inflammatory test.
In addition, HPLC-MS was used to determine the molecular weight of the bee venom melittin enzyme-degrading peptide complex.
As a result, it was found that the protein denaturation can be inhibited even at a low concentration of 40 μg/ml of 50% (IC50) inhibition rate of the peptide complex obtained in this study, and in the cell viability test by MTT assay, no toxicity was observed below 200㎍/㎖ of peptides complex.
In addition, inhibition of β-hexosaminidase secretion also showed that the inhibitory effect on the secretion of β-hexosaminidase induced by a A23187 increased with dose.
In the anti-inflammatory test, cytokine TNF-α derived from TH1 cell and cytokine IL-4 derived from TH2 cell were measured, and the production of inflammatory cytokine was decreased according to the administration concentration.
In ionic curent chromatograms of HPLC-MS analysis, main peaks were observed at 2.4, 2.9, 3.6, and 4.6 minutes.
So, The authors decided to perform molecular weight measurements from 2.0 to 4.7 minutes.
As a result, the following results were obtained.
1). HPLC-MS analysis of the melitin enzyme digesting peptide complex prepared by this study was performed. As a result, there are 4 kinds of free amino acids, 7 kinds of di-peptides, 7 kinds of tri-peptides, 12 kinds of tetra-peptides, 12 kinds of penta-peptides, 9 kinds of hexa-peptides and hepta peptide, 3 kinds of nona-peptide, 3 kinds of deca-peptide, 1 type of hendeca-peptide, 1 kind of dideca-peptide, 3 kinds of trideca-peptide, 4 kinds of tetradeca-peptide, 7 kinds of pentadeca-peptide, 1 kind of hexadeca-peptide, respectively, and the molecular weight of each peptide was specified.
2). The major peaks of the melitin enzyme digestion peptide complexes prepared by this study were 2.4, 2.9, 3.6, and 4.6 minutes, respectively. The main compound molecular weights were 1482, 1556, 1383, 1038, 620, 1569, 607, 973, and high degree oligomers composed with at least 5 or more peptide bonds.
3). Compared to the type of peptide identified by G. Kreil (two heptapeptides, four tetra peptides, one pentapeptide, six types of tripeptide, and three dipeptides), the type of peptide I could see a lot.
In addition, it has been shown that the peptide complexes of melittin, trypsin and chymotrypsin hydrolyzates of G. kreil are composed of low molecular weight peptides, whereas the peptide complexes obtained in this study are composed of oligo peptides with molecular weight of 5 or more amino acids .
4). On the other hand, the enzymatic digestion of G. kreil showed a low molecular peptide complexes of 3-4 peptides on average, whereas the bee melittin enzyme digesting peptide complex in this study showed more than 5 peptides and more than 10 high oligo peptides. By this structure, it is thought that the peptide complex of this study is preserved without losing its physiological activity.
5). The peptides produced by this study inhibited the heat denaturation of albumin protein and inhibited the production of TNF-α and IL-4, which are inflammatory cytokines, without inducing cytotoxicity as a result of MTT assay.
From these results, it was possible to characterize the constituents and molecular weight of the active substance at the molecular level by applying HPLC-MS to a small amount of physiologically active substance which is small in quantity and difficult to be detected by conventional HPLC analysis. The results are expected to be useful as a means of evaluating small amounts of environmental pollutants in the natural world.
주제어
#벌독 HPLC-MS 펩타이드
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