JPS6212998B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for preparing type C cytochromes.
More specifically, the present invention relates to a method for isolating a new heat-resistant cytochrome exhibiting the following physical properties from Thermus thermophilus HB8 cells. a The oxidized form has absorption maxima at 360, 409, 528, and 693 nm, and the reduced form has absorption maxima at 417, 522, 549, and 555 nm. b The millimolar molecular extinction coefficient of the reduced α absorption band (554.8 nm) is 22.0. c The isoelectric point is PH3.8. d The molecular weight is approximately 11,000 as determined by the sedimentation equilibrium method and approximately 10,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Cytochrome is a heme protein whose unique function is to transfer reducing equivalents through redox of heme iron.It mainly works in energy exchange fields such as respiration and photosynthesis, and also plays other roles necessary for living organisms. It is a substance in living organisms that is widely involved in biological phenomena associated with substance production and electron transfer. Regarding the pharmacological effects of cytochrome C, there are reports that it is effective in treating heart disease and gas poisoning.
It is also commercially available as enzyme preparations such as cell metabolism activators and tissue respiration activators. The present inventors conducted a comparative biochemical study on Thermus thermophilus HB8.
We focused on the C-type cytochrome, attempted to isolate it, and arrived at the present invention. That is, the gist of the present invention is a method for preparing C-type cytochrome, which is characterized by crushing the cells of Thermus thermophilus HB8 strain and isolating cytochrome C 555,549 exhibiting the following physical properties from the crushed product. exists in a The oxidized form has absorption maxima at 360, 409, 528, and 693 nm, and the reduced form has absorption maxima at 417, 522, 549, and 555 nm. b The millimolar molecular extinction coefficient of the reduced α absorption band (554.8 nm) is 22.0. c The isoelectric point is PH3.8. d The molecular weight is approximately 11,000 as determined by the sedimentation equilibrium method and approximately 10,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Furthermore, to explain the method of the present invention in detail, the material used in the present invention is Thermus thermophilus HB8 (ATCC27634).
International J. of Systematic Bacteriol.
It is a strain belonging to the genus Thermus described in 24 , 102-112. To isolate C-type cytochrome from the cells of the strain, the cells are crushed and the cells are separated from the crushed material. Specifically, the bacterial strain was first cultured at 75°C for about 6 hours using a medium containing 0.5% peptone, 0.4% yeast extract, 0.2% sodium chloride, and 0.1% glucose, and then harvested at the late logarithmic growth phase. Then, the obtained bacterial cells are disrupted by a conventional method, for example, by sonication. Cytochrome C is then separated from the resulting crushed material by a conventional method. Separation method 1
To explain an example, a Tris-HCl buffer (PH7.8) containing EDTA, magnesium chloride, potassium chloride, and β-mercaptoethanol is added to the crushed material, stirred, and the supernatant obtained by centrifugation at 7500 rpm is further
Centrifuge at 22000g for 15 hours and precipitate (membrane fraction)
Triton X-100 (manufactured by Wako Pure Chemical Industries, Ltd., surfactant, Triton is a trademark) and Tris-HCl buffer (PH7.5) containing potassium chloride are added to the solution, stirred, and centrifuged to obtain a supernatant. Add 0.5% Triton X to the supernatant.
After dialyzing with Tris-HCl buffer (PH8.5) containing 100, add to diethylaminoethyl cellulose column, wash with the same buffer plus 0.05M potassium chloride, and elute with 0.2M potassium chloride. do. The eluate was concentrated by ultrafiltration, dialyzed against 0.05M Tris-HCl buffer (PH8.5), added to a diethylaminoethyl cellulose column, washed with the same buffer plus 0.05M potassium chloride, and further
Elute with 0.2M potassium chloride. After concentrating the eluate by ultrafiltration, it was dialyzed against 0.05M Tris-HCl buffer (PH7.5), and then adsorbed on a diethylaminoethyl cellulose column. The cytochrome fraction was eluted with a concentration gradient of potassium chloride, and this was then filtered again. After concentration by ultrafiltration, it is passed through a Cephadex G-75 column (gel filtration agent, Cephadex is a trademark) to collect a reddish fraction, and when this is subjected to isoelectric focusing, cytochromes are detected at a pH of around 3.8. You can get C -555,549. Cytochrome C -555 obtained in this way,
549, its oxidized form is 360, 409, 528, 693nm,
The reduced form has absorption maxima at 417, 522, 549, and 555 nm, and the millimolar molecular extinction coefficient of the reduced form α absorption band (554.8 nm) is 22.0. The isoelectric point is PH3.8, and the molecular weight is approximately 11,000 by sedimentation equilibrium method and approximately 10,000 by polyacrylamide gel electrophoresis method in the presence of sodium dodecyl sulfate. Example (1) Culture Thermus Thermophilus HB8
(ATCC27634) was cultured using a medium containing 5 g of peptone, 4 g of yeast extract, 1 g of glycol, and 2 g of sodium chloride. That is,
First, the above bacteria were inoculated into two 20 ml test tubes containing about 3 ml of the above medium, and the bacteria were inoculated into two 20 ml test tubes containing about 3 ml of the above medium.
℃) for two days and nights. Then the above medium 1
I planted 8 Sakaguchi Kolben trees with 5 in them.
The culture was incubated overnight at 75°C with shaking. Furthermore, the seeds were planted in four 20 jar fermenters, aerated at 75°C for about 8 hours, and collected at the late stage of logarithmic growth (Cretz number of about 400) (using a shear press type continuous centrifuge), and then cultured in physiological saline. I washed it lightly at about 2. The bacterial cells were stored frozen at -20°C. The bacterial yield is determined by the culture medium.
80, the wet weight was about 700g. (2) Grinding of bacterial cells 500 g (wet weight) of bacterial cells that had been frozen and stored at -20°C was left at room temperature for about 30 minutes, and cut into dice with a knife before the bacterial cells had melted ( (The rest of this procedure was carried out at 4°C.) This was placed in a large mortar, sprinkled with about 500 g of aluminum oxide for grinding, and the bacterial cells were ground mechanically using a pestle. After about 30 minutes, an appropriate amount (about 125 g) of the above aluminum oxide was added, and the grinding was continued for about 1 hour. Once the bacterial cells were crushed and the whole mixture became thick, we moved on to the next extraction operation. (3) Separation of membrane fraction 1mM MEDTA, 10mM magnesium chloride,
50mM Tris-HCl buffer (PH) containing 135mM potassium chloride and 7mM β-mercaptoethanol
Add 7.8) to 1.3 mortar and mix with pestle for about 30 minutes.
I made a book and continued stirring. thick suspension
Centrifugation was performed at 7500 rpm for approximately 45 minutes. Add supernatant
Centrifugation was performed at 15,000 rpm for 15 hours. The membrane fraction was obtained as a precipitate. (4) Solubilization of membrane fraction The obtained precipitate was dissolved in 1mM EDTA, 10mM magnesium chloride, 135mM potassium chloride and 7mM β-
Homogenize with 50mM Tris-HCl buffer (PH7.8) containing mercaptoethanol,
Centrifugation was performed at 30,000 rpm for 2 hours. Add 10% Triton
Homogenization was carried out using Tris-HCl buffer (PH5) at final concentrations of 20 mg/ml, 2%, and 0.05M, respectively. Add potassium chloride to this to make 1M
After stirring overnight, the mixture was centrifuged to obtain a supernatant. (5) Purification of cytochrome C 555,549 The above supernatant contains 0.5% Triton X-100.
Dialyze against 0.05M Tris-HCl buffer (PH8.5),
Equilibrate the dialysate with the same buffer and perform DEAE.
- After adsorbing on a cellulose column (φ5 x 30 cm) and washing with the above buffer containing 0.05M potassium chloride, the concentration of potassium chloride was increased to 0.2M and the eluate was collected. Diaflow membrane UM-2
After concentration by ultrafiltration using a membrane manufactured by Amicon (Diaflow membrane is a trademark),
Dialysis was performed with 0.05M Tris-HCl buffer PH7.5.
DEAE-cellulose column (φ2 x 38 cm) equilibrated with 0.05M Tris-HCl buffer PH7.5
The linear concentration gradient of potassium chloride (0→
0.3M) and 0.05M Tris-HCl buffer PH7.5. A cytochrome fraction was obtained at a potassium chloride concentration of around 0.2M. After concentrating this again by the same ultrafiltration as above, it was passed through a column (φ1.5 x 90 cm) of Cephadex G-75 (gelling agent manufactured by Pharmacia, Cephadex is a trademark), and 10mM
It was developed with potassium phosphate buffer (PH7.0) and the reddish fraction was collected. This was subjected to isoelectric focusing [1% Carrier Ampholyte (PH gradient agent manufactured by LKB, Carrier Ampholyte is a trademark) PH3.5 ~
5.0 for over 48 hours], highly purified cytochrome C -555,549 with a pH of around 3.8 was obtained. The yield is approximately 2.6 per 500g (wet weight) of bacterial cells.
It was mg. (6) Measurement of properties of cytochrome C -55,549 (a) Absorption band of oxidized form Potassium ferricyanide was added to the sample solution and approx.
It was added to a concentration of 10 μM, dialyzed against 10 mM Tris-HCl buffer (PH7.8), and the spectrum was measured. It had absorption maxima at 360, 409, 528, and 693 nm. (b) Absorption band of reduced form A trace amount of hydrosulfite powder was added to the sample in (a) above, and the spectrum was measured.
Absorption maxima were observed at 417, 522, 549, and 555 nm. (c) Millimolar molecular extinction coefficient The millimolar molecular extinction coefficient of the α absorption band (554.8 nm) of the reduced form was measured by the pyridine hemochrome method according to the method described in J.Biol.Chem., 146 , 605 (1942). By the way, it was 22.0. (d) Isoelectric point The isoelectric point was measured according to the method described in Acta Chem.Scand., 20 , 820 (1966), and it was found to be PH3.8. (e) Molecular weight The molecular weight was determined to be 11,000 by the sedimentation equilibrium method according to the method described in Biochemistry, 3 , 297 (1964). The molecular weight was determined to be 10,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate according to the method described in J. Biol. Chem., 244 , 4406 (1969). (7) Thermal stability of cytochrome C -555,549 (1) Effect of temperature on 695nm absorption band: Oxidized cytochrome C in the 600-730nm region
The absorption spectrum of -555,549 is not affected by 1M guanidine hydrochloride at 25℃. The spectral changes in this temperature range were measured in a 1M solution of the denaturing agent.
The 695nm absorption band was not lost even at 80°C.
(Figure 1) The change in absorbance at 693 nm of cytochrome C -555,549 is compared to that of horse cytochrome C at 695 nm.
compared with the change in absorbance at . (Figure 2) These results indicate that cytochrome C -555,549
However, with regard to the bond between heme iron and the sixth ligand methionine, it has been shown to have higher thermal stability than horse cytochrome C, to have its original function even at high temperatures, and to be able to conduct electron transfer. (2) Temperature dependence of circular dichroism at 222 nm The thermal stability of cytochrome C -555,549 is
measured by reduction in circular dichroism at 222 nm in the presence of 1 M guanidine hydrochloride;
The thermal stability of horse cytochrome C was compared.
(Figure 3) That is, as a result of examining the change in circular dichroism (the molar ellipticity) at 222 nm due to temperature in the presence of guanidine hydrochloride, it was found that horse cytochrome C changed its predetermined form at 75°C. Although lost, cytochrome C -555,549
substantially retained its original morphology at 75°C. From this, cytochrome C -555,549 is
It is clear that it is much more thermostable than equine cytochrome C, and is stable even at 75°C. (8) Novelty of cytochrome C -555,549 Cytochrome C -555,549 of the present invention is an endogenous membrane protein and is a well-known cytochrome C ₁ that is not directly oxidized by cytochrome oxidase.
is similar to Cytochrome C ₁ is a type of cytochrome C that was discovered in bovine cardiac muscle by Yakushiji and Okunji (1940), and has been found in the mitochondrial inner membrane of animals, plants, and yeast, as well as in certain types of bacteria. Contains heme C, the reduced form has wavelengths of 553, 523, and 418 nm, and the oxidized form has wavelengths of 410 to 418 nm.
It has an absorption band at 411 nm and can be solubilized and extracted from tissues with surfactants. The minimum molecular weight is approx.
36,000, and in solution it shows a value of about 360,000. Standard redox potential E ₀ = 0.223V, isoelectric point PH3.6, no auto-oxidation, oxidized by cytochrome C oxidation type,
Involved in electron transfer from cytochrome B to cytochrome C. (Refer to page 777 of the second edition of Biological Dictionary.) However, while ordinary cytochrome C ₁ associates without the use of a surfactant, the cytochrome C -555, 549 of the present invention associates without the use of a surfactant. The novel feature is that there is nothing to do. In other words, the well-known molecular weight of bovine myocardial cytochrome C ₁ is approximately 36,000 using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), and approximately 360,000 using sedimentation equilibrium method. The molecular weight of the cytochrome C -555,549 of the present invention is approximately 10,000 by both polyacrylamide gel electrophoresis and sedimentation equilibrium methods, indicating that it does not associate even in the absence of a surfactant. As mentioned above, the cytochrome C of the present invention -
555 and 549, for example, have high heat resistance, substantially retaining their original form even at temperatures as high as 75°C, at which the well-known horse cytochrome loses its form, and it also has a surfactant. It has a characteristic not found in the well-known cytochromes, that is, it does not associate even if it is not present.

[Brief explanation of the drawing]

Figure 1 shows the temperature change in the absorption spectrum of oxidized cytochrome C -555,549 in the presence of guanidine hydrochloride. Cytochrome (0.194mM) contains 1M guanidine hydrochloride
Dissolved in 50mM phosphate buffer (PH7.0). Figure 2 shows 695nm or
It shows the change in absorbance at 693 nm with temperature. In the figure, the mouth is horse cytochrome C, ○ is cytochrome C -555,549, and other conditions are the same as in Figure 1. Figure 3 shows that in the presence of guanidine hydrochloride,
It shows the temperature change of circular dichroism at 222 nm. The opening in the figure is horse cytochrome C, and the ○ is cytochrome C -555, 549 [both in 50mM phosphate buffer containing 1M guanidine hydrochloride (PH7.0)]
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Claims (1)

[Scope of Claims] 1. A method for preparing C-type cytochrome, which comprises crushing the cells of Thermus thermophilus HB8 strain and isolating cytochrome C 555,549 exhibiting the following physical properties from the crushed product. a The oxidized form has absorption maxima at 360, 409, 528, and 693 nm, and the reduced form has absorption maxima at 417, 522, 549, and 555 nm. b The millimolar molecular extinction coefficient of the reduced α absorption band (554.8 nm) is 22.0. c The isoelectric point is PH3.8. d The molecular weight is approximately 11,000 as determined by the sedimentation equilibrium method and approximately 10,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.