JPH07278004A - Production of red blood coagulant factor - Google Patents

Abstract

PURPOSE:To provide a production of a new red blood coagulant originating from a marine plant where its coagulation activity is high and can be controlled by the ionic strength with excellent selectivity in saccharide chain recognition. CONSTITUTION:A red algae, Gracilaria sp., is extracted with a phosphate buffer solution and the extract is salted out to obtain a rough active fraction, then the fraction is subjected to chromatography to separate the fraction of more than 100,000 molecular weight whereby the objective coagulant factor is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to the red algae of the genus Ogonori (G.
racilaria sp. ) Is used as a raw material to produce a novel hemagglutinin having specific properties.

[0002]

2. Description of the Related Art Hemagglutinin, which exhibits specific behavior to erythrocytes of each animal, is widely used as a test reagent or a separation material in the fields of medicine, pharmaceuticals, biochemistry and the like. This hemagglutinin is roughly classified into animal-derived ones and plant-derived ones, but plant-derived ones have been attracting attention in practical use in consideration of availability in large quantities and easy processing. There is.

So far, as the hemagglutinin derived from this plant, concanavalin A (Con A) from jack bean and wheat germ lectin (WGA) from wheat as derived from land plants and [[Journal of Bacterio 32. J. Bacteriol.
Vol. 227-237 (1936)], as a source of marine plants (Gracilaria)
GVAI from Verrucosa), Hypni from Hypnea japonica
n A, B, C and D ["Bret. of the Japanese Society of Scientific Fisheries (Bul.Jap.Soc.Sci.F.
ise. ) ”, Vol. 47, pp. 1079-1084 (1981)] and the like.

[0004] However, although a preparation having a high agglutinating activity can be obtained relatively easily from a plant derived from a land plant, the hemagglutination activity is inhibited by a simple sugar such as a monosaccharide or a disaccharide. Therefore, it has the disadvantage that the recognition sugar chain selectivity is low, and that those derived from marine plants are not inhibited by hemagglutination activity by monosaccharides and disaccharides, but by glycoproteins such as fetuin and asialofetuin. Therefore, it is considered that the recognition sugar chain selectivity is high, but it has the drawback that it is difficult to obtain a preparation with high agglutination activity, and both can control agglutination activity by changing the ionic strength. It has the drawback of not being able to.

[0005]

Under the above circumstances, the present invention can control the agglutination activity by the ionic strength,
The purpose of the present invention is to provide a novel hemagglutinin having excellent recognition sugar chain selectivity and high specific activity.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted various studies on hemagglutinins derived from plants, particularly marine plants, and as a result, have found that the red algae of the genus Gragonar
ia sp. ), Hemagglutinin extracted under specific conditions has high aggregation activity and high recognition saccharide selectivity, and
It was found that the aggregation activity can be controlled by the ionic strength,
The present invention has been completed based on this finding.

That is, according to the present invention, a phosphate buffer extract from red seaweed (Gracilaria sp.) Is salted out to collect a crude active fraction, followed by chromatography to obtain a molecular weight of 100,000. The present invention provides a method for producing hemagglutinin, which comprises separating and collecting the above fractions.

As a raw material in the method of the present invention, red algae of the genus Ogonori are used.
laria verrucosa), tsurcilamo (Gr
acilaria chorda), variants thereof are preferred. These red algae are also found in the cold sea, but especially in the warm sea, and they are distributed in almost all coastal areas in Japan, and they are used as agar extenders and sashimi bites.

According to the method of the present invention, the above red algae raw material is sequentially subjected to (a) a water-soluble fraction extraction step, (b) a crude active fraction fractionation step, and (c) an agglutinin purification step. By applying it, a desired hemagglutinin can be obtained.

Explaining preferred embodiments of each of the above-mentioned steps, first, in the step (a), a buffer solution, for example, a sodium chloride-containing phosphate buffer solution is added to the starting red algae to homogenize it, followed by centrifugation. , A crude extract which is a supernatant is obtained. Next, in the step (b), the above (a)
First, ammonium sulfate is added to the extract obtained in the step so that the final concentration is a saturated solution of about 30 to 40% by weight, salting out in the first step is performed, and the formed precipitate is removed by centrifugation. By this operation, contaminants such as pigments are removed as a precipitate fraction. Then, ammonium sulphate was added to the supernatant obtained by centrifugation to add a saturated solution having a final concentration of about 70% by weight, and salting out was carried out in the second step. The precipitated fraction is redissolved in a buffer solution such as sodium chloride-containing phosphate buffer solution to obtain a crude active fraction.

Finally, in the step (c), the crude active fraction obtained in the step (b) is dialyzed against a buffer solution such as a sodium chloride-containing phosphate buffer solution and then separated by chromatography. , To obtain purified hemagglutinin. At this time, as the chromatography, ion exchange chromatography, gel filtration chromatography or a combination thereof is preferably used.

The hemagglutinin obtained by the method of the present invention has the property of aggregating sheep red blood cells treated with (a) pronase with a novel substance, and this agglutination activity is not inhibited by simple monosaccharides or disaccharides. Inhibited by glycoproteins such as fetuin and asialofetuin, (b) the agglutination activity on rabbit red blood cells changes with ionic strength,
In addition, in (c) the globular protein is used as a standard molecular weight substance, it has a characteristic of being eluted in a fraction corresponding to a molecular weight of 100,000 or more in gel filtration chromatography.

[0013]

EFFECTS OF THE INVENTION The hemagglutinin obtained by the method of the present invention is a novel one derived from red algae, and the agglutination activity can be controlled by ionic strength, the recognition sugar chain selectivity is excellent, and the specific activity is high. It has the characteristics of If this property is utilized, the aggregating activity of agglutinin can be controlled by the ionic strength, so that it can be used as a test reagent or a separation material in the clinical field, medical field, biochemical industry field, etc. it can.

[0014]

The present invention will be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

Example 1 (a) Extraction Step of Water-Soluble Fraction Tsurushiramo (produced by Yoshino River estuary in Tokushima Prefecture) was washed with 0.15M sodium chloride aqueous solution and dried in the sun to obtain a dried product. After homogenizing by adding 700 ml of 0.15 M sodium chloride-containing 100 mM phosphate buffer (pH 6.9) to 100 g of this dried product, the homogenized solution was left standing at 4 ° C. for 6 hours and then centrifuged to obtain a supernatant. A crude extract was obtained.

(B) Fractionation step of crude active fraction Next, ammonium sulfate was added to this crude extract so that the final concentration was 35% by weight, and salting out was carried out in the first step. After the addition of ammonium sulfate was completed, the mixture was allowed to stand at 4 ° C. for 1 hour, and then the generated precipitate was removed by centrifugation.
By this operation, contaminants such as pigments were removed as a precipitate fraction. Then, the supernatant obtained by centrifugation was added to a final concentration of 70% by weight.
Ammonium sulfate was added so that the solution became a saturated solution, and second-stage salting out was performed. After adding ammonium sulfate, 4 ℃
After being left overnight at, the generated precipitate was separated by centrifugation. The separated precipitation fraction was redissolved in a 0.15 M sodium chloride-containing 100 mM phosphate buffer (pH 6.9) to obtain a crude active fraction. The hemagglutination activity of the obtained crude active fraction on rabbit red blood cells was 256 units, the specific activity was 3372.9 units / mg protein, and the activity recovery rate was 62.4%. Here, the unit of the agglutination activity was defined as the reciprocal of the maximum dilution rate of the sample in which the agglutination activity can be detected.
The results are shown in Table 1.

(C) Step of purifying agglutinin Next, the crude active fraction thus obtained was treated with 0.15
100 mM phosphate buffer containing M sodium chloride (pH
After dialysis against 6.9), TSKgel DEAE-5P
Separation was carried out by ion exchange chromatography using W to obtain a purified standard product. The minimum protein concentration of the obtained purified sample showing hemagglutination activity against rabbit red blood cells was 0.8763 μg / ml. From the above results, it is understood that according to the method of the present invention, hemagglutinin derived from red algae can be effectively obtained while maintaining its activity.

The ionic strength dependence of the agglutinating activity on rabbit red blood cells of the purified preparation was examined.
The aggregation activity at a concentration of 5M sodium chloride was 2048 units, while the aggregation activity at a concentration of 0.4M sodium chloride was 8 units. The results are shown in Table 2.

Comparative Example 1 In the fractionation step of the crude active fraction of Example 1- (b), instead of the fractionation treatment by salting out in two steps by addition of ammonium sulfate, fractionation treatment with 50 wt% ethanol [“phytochemistry ( Phytochemistr
y) '' Vol. 27, pp. 2063-2067 (198)
8 years))] was carried out, and a crude active fraction was obtained in the same manner as in Example 1. This crude active fraction had a hemagglutination activity of 4 units against rabbit erythrocytes and a specific activity of 53.4 units / m 2.
The g protein and activity recovery was 5.0%. The results are shown in Table 1.

Comparative Example 2 "Comp. Biochem. Physiol." No. 1
Hemagglutinin from red algae was obtained according to the method described in Vol. 02B, pp. 445-449 (1992). The resulting crude active fraction had a hemagglutination activity of 16 units, a specific activity of 149.5 units / mg protein, and an activity recovery rate of 19.5%. The results are shown in Table 1. In addition, the minimum protein concentration showing the hemagglutination activity of the purified preparation against rabbit red blood cells was 32.6 μg / ml, which was equivalent to the specific activity of about 1/40 of Example 1.

Comparative Example 3 From red algae, “Comp. Biochem. Physio
l. 102B, 445-449 (199
2 years) molecular weight of about 50,000 purified according to the method described
Regarding the agglutinin of 0, the ion concentration dependence of the agglutination activity on rabbit erythrocytes was examined. The aggregation activity at both 0.15 M sodium chloride concentration and 0.4 M sodium chloride concentration was 1024 units, and the ionic strength dependence of the aggregation activity was not seen. The results are shown in Table 2.

Comparative Example 4 25 mg of Con A [manufactured by Wako Pure Chemical Industries, Ltd.] was dissolved in 100 ml of phosphate buffer, and the ionic strength dependence of the hemagglutination activity on rabbit red blood cells was examined. The aggregation activity at both 0.15 M sodium chloride concentration and 0.4 M sodium chloride concentration was 64 units, and the ionic strength dependence of the aggregation activity was not observed. The results are shown in Table 2.

[0023]

[Table 1] Note 1) The agglutination activity was calculated from the maximum dilution rate showing agglutination activity by serially diluting the crude active fraction. 2) The activity recovery rate was calculated with the agglutinating activity of the entire crude extract as 100%.

[0024]

[Table 2] Note 1) Agglutination activity was calculated from the maximum dilution rate showing agglutination activity after serially diluting purified agglutinin.

As is clear from Table 1, the crude active fractions of Examples have higher aggregation activity, specific activity and activity recovery rate than those of Comparative Examples 1 and 2, and the activity recovery rates are comparative examples. 1
About 12 times, about 3 times that of Comparative Example 2, and the specific activity is about 63 times that of Comparative Example 1 and about 23 times that of Comparative Example 2. Further, from Table 2, the purified agglutinin of the Example is different from those of Comparative Examples 3 and 4,
It can be seen that the agglutination activity on rabbit red blood cells is controlled by ionic strength.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Lee Tadatomi 2-3-3 Hananomiya-cho, Takamatsu-shi, Kagawa Prefecture Shikoku Institute of Industrial Technology, Institute of Industrial Technology (72) Inventor Hiroshi Uejima Hananomiya-cho, Takamatsu-shi, Kagawa 3-3, Shikoku Institute of Industrial Technology, Institute of Industrial Technology

Claims (2)

[Claims] 1. A red alga, Gracilari
a sp. The salt solution of the phosphate buffer extract from the above) is subjected to salting out to collect a crude active fraction, and then a fraction having a molecular weight of 100,000 or more is separated and collected by chromatography to collect the hemagglutinin. Manufacturing method. 2. The red alga of the genus Ogonori belongs to
2. The production method according to claim 1, which is ilaria verrucosa) or tsurcilamo (Gracilaria chorda) or a subspecies thereof.