JPS61197596A - Straight-chain peptide having reducing activity and production thereof - Google Patents

Abstract

NEW MATERIAL:The straight-chain peptide of formula (n is 1-3). EXAMPLE:gamma-L-glutamyl-L-cysteinyl-gamma-glutamyl-L-cysteinylglycine. USE:Antidote for heavy metals such as Cd, Cu, etc., harmful to human body, and a reducing agent for harmful oxygen radical generated by ionizing radiation. It is free from toxicity and side effect, and is producible easily and quickly at a low cost. PREPARATION:A yeast (e.g. Schizosaccharomyces pombe) is cultured in a liquid medium containing preferably 0.02-2m-mol concentration of a salt of a transition heavy metal element (e.g. cadmium chloride) at 25-30 deg.C for 10-24hr under aerobic condition usually by shaking, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel linear peptide having a reducing action and a method for producing the same.

(Prior Art) It is well known that heavy metal elements such as cadmium, which are taken into the human body through oral ingestion, cause various disorders. Furthermore, when radiation therapy is applied to the human body, it is known that the irradiation of radiation induces radical reactions within the human body, producing harmful oxygen radicals.

Therefore, in the human body, active thiol group (-8H)
It is thought that the cysteine having the above-mentioned properties alleviates the above-mentioned hindrance to some extent by bonding with the above-mentioned heavy metal elements or by reducing and inactivating the above-mentioned active radicals through a redox reaction.

(Problem to be solved) However, the thiol group of the above-mentioned cysteine is easily oxidized in the human body, and most of the disulfide bonds (-8-8-) between the thiol groups Since the formation of L-cystine results in inactive L-cystine, the ability to bind to the heavy metal elements and the ability to reduce radicals cannot be expected in practice.

Therefore, the present invention can maintain high activity in the human body as a chelating agent that binds to heavy metals and inactivates them, and as a radical reducing agent, and does not cause Q-level side effects or toxicity when administered to the human body. The technical problem to be solved is to provide a new substance and a method for its production.

During the search for biological substances that neutralize heavy metal toxicity, the present inventor discovered that a low molecular weight peptide that binds cadmium was synthesized in yeast by induction, and completed the present invention.

(Means for solving problems) The technical means to solve the above problem is the chemical structural formula (In the formula, n represents any integer from 1 to 3.

), and by culturing yeast in a medium containing a salt of a transition group heavy metal element, the linear peptide is induced as a complex compound with the transition group heavy metal element. It is to synthesize.

The linear peptide of the present invention includes γ-L-glutamyl-L-cysteinyl-γ-L, which is a condensation of five amino acids.
- Glutamyl - one cysteinylglycine and 7 amino acids condensed together.
γ-L-glutamyl-cysteinylglycine is a condensation of 1-glutamyl-L to cysteinylglycine and 9 amino acids.
It contains three types of peptides: -L-glutamyl-cysteinyl-γ-monoglutamyl-L-cysteinylglycine. These linear peptides are hereinafter collectively referred to as Kabuistin, and the one consisting of the above seven amino acids is referred to as Kabuistin Δ, and the one consisting of five amino acids is referred to as Kabuistin B.

In addition, by the production method of the present invention, there is a possibility that a peptide in which n is an integer of 4 or more in the chemical structural formula described above can also be synthesized by induction.

Next, as the yeast used in the production method of the present invention, in particular, fission enzyme fJJ (Schizosaccharomyc
es pombe), but other species of yeast, such as saccharomyces cerevisiae
Brewer's yeast, baker's yeast, etc. of the genus Candida, and feed yeast of the genus Candida may also be used. Furthermore, since the guided synthesis of heavy metal-binding proteins or peptides is considered to be a defense reaction universally recognized in living organisms, the linear peptide of the present invention can be induced using microorganisms other than yeast. It is thought that it is possible to synthesize it.

The medium may be one commonly used for culturing yeast, such as a nutrient medium using peptone or the like, or a synthetic medium with a known composition. In addition, from an agar medium with no fluidity,
The use of a liquid medium is advantageous in terms of increasing the yield of kabuistin because it promotes the reproduction of fission yeast and the induced synthesis of kabuistin.

A salt of a transition group heavy metal element is added to the medium.

Salts of transition group heavy metal elements include cadmium, copper,
Salts with highly dissociable materials such as zinc, lead, and mercury, such as salts with non-sulfuric acid, are used. Less dissociated salts such as cadmium phosphate are not effective. As an example of the most effective salts, mention may be made of cadmium chloride, CdCl. The amount of transition group heavy metal elements added varies depending on the type of heavy metal element, but
The concentration is about 0.02 to 2 mmol relative to the medium.

If the amount added is less than the above range, the induced synthesis of Kabuistin will not be carried out, and if it is more than the above range, yeast growth will be poor, which is not preferable. Yeast culture is as usual, about 2
It is carried out under aerobic conditions such as shaking culture at 5 to 35°C. The culture time is about 10 to 24 hours.

Examples of possible structural models of the complex compound of Kabuistin and a transition group heavy metal element obtained by the production method of the present invention include those shown in FIG. 1 or FIG. 2. In FIGS. 1 and 2, the thick line indicates Kabuistin A, and cadmium is shown as a transition group heavy metal element.

Incidentally, it is thought that Kabuistin B1 or the above-mentioned linear peptide composed of nine amino acids also forms a complex compound in a similar bonding manner.

Regardless of their structure, when these complex compounds are adjusted to p112 under strong acidity, for example with hydrochloric acid, they dissociate to produce kabuistin in a free state. For example, it can be isolated using a molecular sieve column that fractionates by molecular weight.

(Function) Kabuistin contains at least two cysteines, and each of the amino groups of these cysteines forms a peptide bond with the carboxyl group at the γ-position of monoglutamic acid. Although not shown in the structural formula, the oxygen atom of the peptide bond forms a hydrogen bond with the thiol group of L-cysteine due to its spatial positional relationship, so the thiol group However, it is significantly more stabilized than the cysteine that makes up the commonly known peptide or the thiol group in free cysteine.

An example of the detoxification effect of Kabuistin against transition group heavy metal elements based on such characteristics is shown in Table 1 and FIG. 3 below. As shown in Figure 3, the activity of LR8 (leucine transfer RNA synthetase), which is an enzyme that binds leucine to leucine transfer RNA, is inhibited by cadmium, but kabuistin was added to the reaction system before adding LR8. In addition, as shown in Table 1, LR
The inhibitory effect of cadmium on 8 disappears. Furthermore, Table 1
In the above, leucine-tRNA refers to production 1 of leucine-bound leucine transfer RNA, which indicates the activity of LR3. Also, in the same table, [beforeEJ [
After E and J indicate the cases where Kabuistin was added before and after the addition of LR8, respectively, and Kabuistin in Table 1 refers to a mixture of Kabuistin A and Kabuistin B (3:2).

(continued on next page) Table 1 None 23.6 100+CdCl28
．． 3μM 9.6 4
3.1 + CdCl, 8.3 μM + Kabuistin 15 μM 2
5.1 106.2 (before
eE) +CdCl,, 8.3μM 15μM
19.0 80.4 (after
r E) +Kabuistin 15μM 2
4.6 103.8 From Table 1, it is clear that kabuistin binds to cadmium, thereby blocking the inhibitory effect of cadmium on enzyme activity.

Next, the results of examining the stability of the complex compound of Kabuistin and a heavy metal element formed in this manner are shown in the fourth column. The figure shows the degree of dissociation of a complex compound of kabuistin and cadmium under hydrochloric acid acidity at 250 nanometers (
In the figure, BPl, BP2. The graphs of GSH and MT-1 are for substances related to the structural model shown in Figure 1 (hereinafter referred to as
BP1), a substance related to the 18-model model shown in FIG. 2 (hereinafter referred to as BP2), a complex compound of glutathione and cadmium, and a complex compound of mouse metallothionein-1 and cadmium. The latter two substances are natural peptides known to have heavy metal binding activity similar to Kabuistin. According to this result, BPI.

BP2 is significantly more stable than complex compounds of glutathione.
Moreover, it does not dissociate at all near neutral pH, which is the pH of the human body, and in particular, it has been shown that the stability of BPl is equivalent to that of mouse metallothionein-1, which has about 10 times the molecular weight.
Moreover, even when the force distin administered to the human body forms complex compounds with cadmium, they are considered to be extremely stable.

Furthermore, the affinity of Kabuistin for cadmium suggests its strong reducing action, so in order to confirm this, we measured the redox potential of Kabuistin, and the results shown in Table 2 were obtained. Obtained.

Table 2 Measurement Substance Redox potential L-cysteine -242 mV Glutatiglutamic acid thione 84 mV Cadistin 8-33 111v Cadistein 3 -314 mV The above measurement is p
This was carried out in a 50 mmolar sodium-phosphate buffer of H7.0 at 20° C. using a Michaelis bath by titration with potassium ferricyanide as an oxidizing agent. Incidentally, the results of titration using sodium hydrosulfite as a reducing agent under the same conditions also showed similar values. Kabuistin A shown in Table 2
The high negative potential of , B indicates that kabuistin has a significantly stronger reducing effect than glutathione.

The above-mentioned affinity of Kabuistin for transition group metal elements and its strong reducing action are brought about by the presence of the aforementioned thiol groups stabilized by hydrogen bonds. It has a detoxifying effect on heavy metal elements such as copper, zinc, lead, and mercury, and its reducing effect prevents the generation of oxygen radicals that are harmful to the human body due to radical reactions induced by radiation exposure to the human body. be.

Next, Kabuistine A and B are both derivatives of glutathione in their chemical structure, and are composed of glycine, L-glutamic acid, and L-cysteine, which are amino acids found in the human body, and are synthesized by induction in living organisms. Since it is a substance that is used in the human body, it is considered that it does not exhibit toxicity or harmful side effects to the human body.

Next, the linear peptide of the present invention can be produced simply, rapidly, and at low cost because it can be synthesized by administering a simple inorganic compound to microorganisms without using organic chemical synthesis means.

(Example) Next, one embodiment of the present invention will be described.

Fission yeast (Schizosaccharomyces)
pombc, this strain is readily available to those skilled in the art. ) was cultured with shaking at 29°C in a liquid medium containing 1 mmolar cadmium chloride cdCI2 in a flask plugged with cotton. The medium was 1% yeast extract <v' (dry powder of mother extract), 2% polypeptone (a product of pepsin-induced degradation of meat), and 2% glucose dissolved in water.

Fission yeast continued to proliferate until about 20 hours had elapsed, and the amount of cadmium taken up reached approximately 5 μg per cell in 1 d of medium.

Bacteria were collected over time. The bacterial collection method was to precipitate the W9 mother by centrifugation and separate it from the liquid medium.

Next, the collected yeast was ground in a mixture of 50 mmolar Tris-HCl buffer (pH 7.6) and 0.1 molar potassium chloride, and the insoluble residue was removed by centrifugation to prepare a yeast extract. .

This extract was applied to a molecular sieve column (Sephadex G-
50>, most of the cadmium has a molecular length of about 4
100 and at a molecular weight of approximately 1700, these materials were analyzed using the method described below.

Specifically, the former and latter substances were measured for cadmium by atomic absorption spectrometry, peptide quantification and amino acid analysis by the known so-called Kjelder-Nessler method, and it was found that the former substance contained four unit peptides and six cadmium units. , the latter substance has two unit peptides and two
Each molecule contained cadmium. or,
Since there was knowledge that these substances seem to contain sulfur,
Hydrochloric acid is added to an aqueous solution of these substances to dissociate the sulfur, which is then absorbed into zinc hydroxide to form zinc sulfide.
This was mixed with chloroform, and methylene blue was reduced by the zinc sulfide extracted into the chloroform layer. Then, when sulfur was quantified by the change in absorption maximum in J3 in spectroscopic analysis of methylene blue, it was found that the former substance was 1
It was found that the molecule contained one unstable sulfur element. As a result, the former substance is BPl shown in the structural model of Figure 1.
It was found that the latter substance was BP2 shown in the structural model of FIG.

Next, in order to understand the structure of the unit peptides of BPl and BP2, a dansyl group was added to the amino terminal amino acid of the peptide, followed by acid hydrolysis and reversed phase high performance liquid chromatography (hereinafter abbreviated as HPLC). A method for detecting amino acids dansylated by glutamic acid, a method for detecting the released amino acids by HPLC after digesting a unit peptide with a carboxypeptidase that releases amino acids at the carboxyl terminal of the peptide, and a method for detecting free carboxyl groups in glutamic acid. (by aminoethylating the peptide and acid-hydrolyzing the peptide) The peptides treated with this method were decomposed with carboxypeptidase, and the amino acid sequence and structure were analyzed by HPLC using a method that allows us to determine the types and composition ratios of amino acids.

As a result, the unit peptides of BPl and BP2 are the same substance,
That is, the above-mentioned Kabuistin A, and ' BPl,
It was found that BP2 also contained a small amount of the above-mentioned Kabuistin B.

Therefore, in order to confirm the structures of Kabuistin A and B, peptides with the estimated structures were synthesized by the method shown in FIG. This method is known as a so-called stepwise organic synthesis method for peptides, and is a conventional organic synthesis method in which amino acids constituting a linear peptide are sequentially condensed, starting from the carboxyl terminal, while protecting a predetermined reactive group. It is a chemical synthesis method. In the figure, 0BZL is a benzyl group that protects the carboxyl group at the α position, BOC is a butyloxycarbonyl group that protects the amine group, MOB is a paramethoxybenzyl group that protects the thiol group, and ■r is the amino group of L-glutamic acid. Protected trityl group (triphenylmethyl group)
■ in the figure represents 3 of trifluoroacetic acid and anisole.
= 1 mixture of each amino acid or peptide at 0℃
After 10 minutes of dissolving the product, add Hekylin to precipitate the product. (■ in the figure) is after dissolving the precipitate in a mixture of dichlorohexyl carposiimide and dichloromethane.
Drying operation, ■ in the figure is Prot in the same figure, respectively.
Kadistine A and Kadistine B with protective groups, shown as ectedl and Protected2, were hydrolyzed in a solution of hydrofluoric acid to preserve 1
5J3: After removing, Kadistine A is extracted with ether.
, shows the operation for isolating Kadistin B.

Then, in division 8911, Xg synthesized cadistinΔ
We compared circular dichroism spectra, reversed-phase high-performance chromatography, and nuclear magnetic resonance spectra for protons in molecules of Kadistine B and the corresponding organically synthesized Kadistine A and Kadistine B. According to the results shown in FIGS. 6 to 10, it was confirmed that the same substances produced the same substance. In addition, in each figure, "Synthesis C
dΔ”.

[The graphs shown as synthetic CdBJ, r natural CdAJ, and r natural CdBJ are graphs of organically synthesized cadistin A, cadistin A induced and synthesized in cadistin B2 fission yeast, and cadistin B, respectively. Figures 6 and 7 show the circular dichroism spectrum (
CD spectrum), the horizontal axis represents wavelength in units of nanometers, and [θ] on the vertical axis represents optical activity for converting linearly polarized light into elliptical polarized light. Furthermore, Figure 8 shows reverse phase high performance chromatography, in which the CdA and CdB peaks are synthesized CdA.
, natural CdA, synthetic CdB, and natural CdB, respectively. Next, Fig. 9(a)(b), Fig. 10(a)
(b) are synthetic CdA, natural CdA and synthetic CdB, respectively.
, which compares the nuclear magnetic resonance spectra of protons of natural CdB, and the horizontal axis in the figure shows the so-called chemical shift in parts per million (ppn+). Also, in Fig. 9(b) and Fig. 10(b), i'"imp
The peak indicated by J is BPI or Br3 from the above-mentioned fission yeast.
This peak is due to the Tris-HCl buffer used for extraction remaining as an impurity.

Next, regarding the physical properties of Kadistine A and Kadistine B, when these substances are obtained as pure substances, their thiol groups form disulfide bonds between molecules and polymerize, making it difficult to measure their physical properties accurately. Therefore, when measured with the thiol group protected by a paramethoxybenzyl group, Kabuistin A has a melting point of 131°C and a D of sodium in a 1°59/1rdl aqueous solution of trichloromethane.
Kabuistin B had a melting point of 107°C and an optical rotation of -1.05° as measured by a line.

(Effect) The linear peptide of the present invention can be utilized as a novel heavy metal antidote and a prognostic agent for radiation irradiation. It can also be used to collect heavy metals in wastewater treatment.

Moreover, the above-mentioned linear peptide can be produced easily, rapidly, and inexpensively by the production method of the present invention.

[Brief explanation of drawings]

Figures 1 and 2 are explanatory diagrams showing the bonding state of a possible structural model of a complex compound of kabuistin and cadmium, Figure 3 is a graph showing inhibition of enzyme LR3 activity by administration of cadmium chloride, and Figure 4 is a graph showing the inhibition of enzyme LR3 activity by administration of cadmium chloride. The degree of dissociation in each 11H of the complex compound of kabuistin and the control substance with cadmium,
A graph showing the chemical luminosity, Figure 5 is a schematic diagram showing the organic chemical synthesis method of Kabuistin, and Figures 6 and 7 compare the circular dichroism spectra of synthetic and natural CdA and CdB. The graph shown in Figure 8 is a graph showing Kabuistin's reverse phase high performance chromatography, and Figure 9 (a
)(b) and FIG. 10 (a>(b) are graphs showing comparative nuclear magnetic resonance spectra of synthetic and natural CdA and CdB, respectively.

Claims (6)

[Claims] (1) Chemical structural formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, n represents any integer from 1 to 3. ) is a linear peptide indicated by (2) γ-L-glutamyl-L-cysteinyl-γ-L
The linear peptide according to claim 1, which is -glutamyl-L-cysteinylglycine. (3) γ-L-glutamyl-L-cysteinyl-γ-L
-Glutamyl-L-cysteinyl-γ-L-glutamyl-L-cysteinylglycine
Linear peptides as described in Section. (4) By culturing yeast in a medium containing salts of transition group heavy metal elements, chemical structural formulas ▲mathematical formulas, chemical formulas, tables, etc.▼ (where n represents any integer from 1 to 3) .) A method for producing a linear peptide, which comprises induction synthesizing the linear peptide represented by the following formula as a complex compound with the transition group heavy metal element. (5) Yeast is fission yeast (Schizosaccharo)
5. The method for producing a linear peptide according to claim 4, wherein the linear peptide is S. myces pombe. (6) The method for producing a linear peptide according to claim 4, wherein the transition group heavy metal element is cadmium.