卵蛋白に関する研究 V : 孵卵中の卵白組成蛋白質の変化について(農芸化学部門)

孵卵中に起る卵白組成蛋白質の消長をC. M. C.カラムクマトグラフィーにより追跡し以下の結果を得た。卵白は孵卵中胚に吸収されてその量を減じて行き, 孵卵開始後15日目位で分離不可能となる。孵卵6日目頃から, それまで卵白中に存在しなかつたAnionic proteinが出現する。Ovomucoid fractionは6日目頃から全卵白蛋白量に対する比率が増加する。Ovalbuminは孵卵の進行と共にA_1に対するA_2+A_3の比率が増加する。Conalbuminは孵卵の進行と共に減少しlysozymeは増加する。By means of fibrous CMC-Column chromatography the relative changes in individual egg white proteins of fertilized hen\u27s egg during the course of incubation, were studied. The results summarized were as follows. 1. The egg white was absorbed by embryo and decreasing its quantity during incubation. Finally after two weeks of incubation, the most of egg white was absobed and could not separate it. 2. On the 6th day from the start of incubation, the anionic protein was appeared which has not been existed in the egg white originally. 3. Ovomucoid fraction was increased after 6th day of incubation 4. Ratio of A_2+A_3/A_1 in the ovalbumin fraction was increased along with the proceeding of incubation. 5. Conalbumin was decreased and lysozyme was increased during the incubation

卵蛋白に関する研究 (III) : 繊維状 CMC の調製と卵白 flavoprotein の分別

(1)蛋白質のカラムクロマト用の新しい繊維状CMCを特殊な方法で調製し, そのD.S., 性質, 滴定曲線, 並びに蛋白吸着能を調べ, 極めて優秀なcation exchangerであることを認めた。(2)繊維状CMC 1g当りの平均蛋白吸着量は約0.5gで, その吸着能力は驚異的で, 而も吸着蛋白の溶出回収率は93%以上で蛋白及び酵素の調製, 精製に極めて便利であることを実証した。(3)卵白の新発見flavoproteinを繊維状CMCによつて分別して, その蛋白部分が2成分からなることを確認し, このflavoproteinのapoproteinについて考察した。1. The authors prepared a fibrous carboxymethyl cellulose (CMC) cation exchanger, the new adsorbent for column chromatography of proteins, by our own method, and investigated the degree of substition (D.S.), physical properties, titration curves and protein adsorption capacity of the adsorbent. It was shown that this fibrous CMC possess high capacity for the adsorption of protein, and was excellent cation exchanging adsorbent for column chromatography of proteins. 2. The average amount of protein adsorbed by this adsorbent was 0.5g of protein per 1g of fibrous CMC. It is very high adsorption capacity, moreover recovery of adsorbed protein on the column in effluent solution exceeded 93%, and this adsorbent will be very useful in preparation and purification of proteins or enzymes. 3. By the chromatographic resolution of newly found flavoprotein of egg white on the fibrous CMC column, it was confirmed that protein moiety of this flavoprotein consist of two components. The apo-proteins of this flavoprotein were discussed

卵蛋白に関する研究 IV : 卵白新フラビン蛋白について(農芸化学部門)

卵白から新しいFlavoproteinを約0.8%の収量で得, そのflavin部分はfree riboflavinであることを認めた。卵白flavoproteinはovomucoid fraction中に集中して存在することを確認した。ovomucoid fractionはpulp状CMC column chromatography及びelectrophoresisによつてI∿Vの5成分から成ることを確認した。M. B. Rhodesらのplate testを改良した筆者らの独特の方法でovomucoidのantitryptic activityを測定して, ovomucoid 5成分のうちantitryptic activityはcomponent I, II, III, の3成分蛋白中にあり, component IIIがその主要部分であることがわかると同時にIV, V componentにはantitryptic activityが全然なかつた。ovomucoidのantitryptic activityは熱に対して可成り安定で90°20分の熱処理は活性に全然影響を与えず, 100°5分の熱処理によつてその活性は3/4に低下した。ovomucoid 5成分のうちIV, V, componentとfree riboflavinとが結合したものが卵白中の新しいFlavoproteinであることがわかり, これに対して筆者らはFlavomucoid K_1及びK_2と命名した。1 The new flavoproteins have been obtained from egg white and yield of this protein was 0.8% of whole egg white protein and it recognized that flavin moiety of this flavoprotein is free riboflavin. 2 It is confirmed by its physico-chemical properties, that the new flavorotein is concentrated in the ovomucoid fraction. 3 By means of fibrous C. M. C. column chromatography, and electrophoresis, it has been confirmed that the ovomucoid fraction is consist of five components. 4 Antitryptic activities of ovomucoid fraction have been determined by the authors method which modified the spot plate test of M. B. Rhodes et al, it is found that antitryptic activities have been existed in the component I, II and III, and component III protein is main component of antitryptic activity of ovomucoid while component IV and V have no activity. 5 The antitryptic activity of ovomucoid is not effected by heat treatment at 90℃ for 20 minutes but by the treatment at 100℃ for 5 minutes, its activity has been reduced to 3/4 of original activity. 6 The authors recognized that the newly found flavoprotein of egg white is composed of IV and V component of ovomucoid and free riboflavin. The authors named these two kinds of new flavoproteins as flavomucoid K_1 and K_2 respectively

各種保存条件下での牛乳κ-カゼインの変化(農芸化学部門)

UHT滅菌乳の長期貯蔵による沈殿生成やゲル化の機構を解明する予備実験として, 牛乳κ-カゼインの各種保存条件下における変化を, 安定化能, 4.5M尿素を含むディスクおよびSDS-ポリアクリルアミドゲル電気泳動により検討した。保存条件としては, 温度-20,4,25℃で, 溶液(70mM KCIを含む10mMイミダゾール塩酸緩衝液pH7.1,4.5M尿素, 蒸留水, 濃度はいずれも10mg/ml)中または, 凍結乾燥標品の12種を設定し, 1,3,5,7ケ月間の保存状態を検討した。-20℃では, ほとんど変化せず, いずれも保存状態は良好であった。4℃では, 4.5M尿素中および凍結乾燥品での保存が良かった。また, 25℃では, 凍結乾燥保存を除き, 全般的に保存性は悪かった。両電気泳動の結果から, 長期保存によってκ-カゼインは高分子化や分解が進んでいることが明らかとなり, 同時に, それらの進行と安定化能の低下との間に相関が認められた。The formation of precipitate and the gelation of ultra high-temperature sterilized milk have been observed during the long storage. As the preliminaly experiments to make clear the mechanism of these phenomena, the effects of various preservative conditions on bovine κ-casein were investigated by the stabilizing ability test for α_-casein in the presence of calcium ion, disc gel electrophoresis containing 4.5M urea and sodium dodecylsufate (SDS)-polyacrylamide gel electrophoresis. κ-Casein was stored at -20°, 4°and 25℃ as the solution dissolved in 10mM imidazole-HCl-70mM KCl buffer (pH7.1), 4.5M urea and distilled water and as the lyophilized material for 1,3,5 and 7 months. When κ-casein was stored at -20℃ at various states, the stabilizing ability was maintained and the behaviors on both electrophoreses were not different from those of native one, though the samples at 25℃ lost the ability quickly. The preservation at 4℃ in 4.5M urea solution did not affect on the properties of κ-casein. The lyophilized material was stable at every tested temperatures through the experimental period. It was considered that the loss of stablizing ability of κ-casein was due to the polymerization and the degradation from the results of disc and SDS gel electrophoreses

ペプシン処理分離大豆たん白質食のラット肝機能および胆汁酸濃度への影響(農芸化学部門)

植物性たん白質(主に大豆たん白質)は動物性たん白質(主にカゼイン)に比べて, 血清コレステロール濃度低下作用を有していることが知られている。著者らは先に, 分離大豆たん白質をペプシンで限定水解することによって, その作用を強めることを発見した。大豆たん白質の血清コレステロール濃度低下作用メカニズムを解明するため, コレステロール代謝を考慮すると, 肝臓および小腸の機能を検討することが重要である。本研究は, 分離大豆たん白質食, ペプシン処理分離大豆たん白質食および市販固型食を用いてラットを飼育し, 食餌たん白質のラット肝機能におよぼす影響を比較検討し, 更に, 肝臓および小腸での胆汁酸濃度, 血清遊離アミノ酸パターンを測定した。肝機能を推定するため, 血清中のGlutamate Oxaloacetate Transaminase, Glutamate Pyruvate Transaminase, Lactate Dehydrogenase, Leucine Aminopeptidase, Alkaline Phosphataseの活性を測定した。その結果, 供試食餌たん白質では肝細胞の破壊や胆汁流出障害は観察されなかった。また, 肝内胆汁酸濃度と血清コレステロール濃度とは比例していたが, 小腸胆汁酸濃度には相関がなかった。すなわち, 血清コレステロール濃度調節機構の解明には, 肝機能を更に検討することの必要性が示された。また, トレオニンが, コレステロール代謝と最も関連深いアミノ酸であることが明らかにされた。It is well-known that vegetable protein (usually soybean protein) has hypocholesterolemic effect compared with animal protein (usually casein). In our previous papers, we indicated that pepsin-treated soybean protein isolate was more hypochole-sterolemic than intact soybean protein isolate. It is important to examine the functions of liver and small intestine in order to make clear the mechanism of hypocholesterolemic effect by dietary soybean protein considering from the point of view of cholesterol metabolism and catabolism. In this paper, effects of dietary protein on liver function and bile acids concentration in liver and small intestine were examined. Rats were fed with soybean protein isolate diet, pepsin-treated soybean protein diet or commercial diet. Serum enzyme acitivities were measured to evaluate the liver function of rats fed various protein diets. Enzymes measured were glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase, lactate dehydrogenase, leucine aminopeptidase and alkaline phosphatase. In the case of rats fed those diets, the destruction of liver cell and the disease of biliary flow were not observed. Serum cholesterol level was proportion to the bile acids concentration in liver, but not in small intestine. Obtained results suggest the necessity of further examinations of liver function in order to clarify the regulation mechanism of serum cholesterol level by dietary protein. It is indicated from serum aminogram that threonine is closely related to cholesterol mechanism

クロレラからの血圧降下作用糖蛋白質の検索(農芸化学部門)

クロレラから, 血圧降下作用物質が部分精製された。生クロレラの細胞壁破砕物を冷アセトンにて脱脂脱色後, 冷水抽出物が得られた。冷水抽出物の80%エタノール不溶性画分に降圧作用が認められた。80%エタノール不溶性画分のBio-Gel P-60ゲルロ過, DEAE-セルロースクロマトグラフィーにより, ほぼ均一な降圧画分D-IIが得られた。画分D-IIの分子量は, 約80,000と推定された。画分D-IIを, 13週令の血圧180∿190mmHgのSHRに0.5mg/100g体重の割合で腹腔内投与すると, 1時間後に140∿160mmHgという血圧になり, 強い降圧効果が認められた。画分D-IIは, ガラクトース, マンノース, グルコサミンを含む糖蛋白質で, アスパラギン酸, グルタミン酸, アラニン, プロリンの含量が高く, 塩基性アミノ酸が少なかった。The antihypertensive material was partially purified from chlorella. Cell wall of chlorella was destroyed with Dyno-mill. After defatted and decolored with cold aceton, water extract was obtained from chlorella by the extraction at 4℃ for 24 hours. 80% ethanol precipitate from water extract was fractionated by Bio-Gel P-60 gel filtration followed by DEAE-cellulose chromatatography. About 50mg of antihypertensive fraction D-II were obtained from 10g of chlorella. The molecular weight of fraction D-II was about 80,000 dalton. The blood pressure of spontaneously hypertensive rat (13 weeks of age) was decreased from 185 to 150mmHg, when fraction D-II was injected intraperitoneally at the concentration of 0.5mg/100g of body weight. Fraction D-II contained galactose, mannose and glucosamine. The contents of aspartic acid, glutamic acid, alanine and proline in fraction D-II were large

鶏卵白中のシアル酸に関する研究(農芸化学部門)

鶏卵白から硫安分別法によって調製した粗オボムチンから, N-acetylneuraminic acidを単離, 同定する研究を行なった。粗オボムチンから結合型シアル酸を遊離させるための, 加水分解条件を検討した結果, 0.1N硫酸, 80℃, 1時間の条件が最も効果的で, 加水分解回数も2度行なえば充分であることが確認された。この条件以上の硫酸濃度および温度では, かなりのシアル酸の分解がおこることが認められた。シアル酸の分離精製に用いたDowex 1(HCOO-型)樹脂の再生方法を検討し, 能率の良い簡易法を見出した。従来の再生方法とこの簡易法の併用により, 短時間で樹脂の再生を行なうことができた。粗オボムチンから, 白色針状結晶のシアル酸を得たが, 操作中の損失は最大限15.7%であり, 粗オボムチン中のシアル酸含量は, アルカリTBA法で測定して, 約0.5%であった。この結晶シアル酸は, すべての比色定量法に陽性であり, ペーパークロマトグラフィー, 赤外吸収スペクトル, X線回折スペクトルをみたところ, N-acetylneuraminic acidの標準品と全く一致した。過沃素酸消費量は, シアル酸1モル当り5モル, フォルムアルデヒド生成量はシアル酸1モル当り1モル, アセチル基をシアル酸の1モル当り1モルを含み, グリコール基は検出されなかった。The sialic acid, N-acetylneuraminic acid, was isolated from the crude ovomucin fraction of hen\u27s egg white which contained about0.5 per cent sialic acid, and the yield of the purified sialic acid from ovomucin was about 84.3 per cent. To isolate the sialic acid from ovomucin, a range of hydrolysis and purification procedures were examined, and it was confirmed that the most effective hydrolytic condition to liberate the sialic acid in the free state, was heating at 80℃ for 1 hour with 0.1N sulfuric acid. The hydrolysis had to be repeated twice to liberate 98 per cent of total sialic acid. The sialic acid obtained was identified as N-acetylneuraminic acid, by means of paper chromatography, six colour reaction, infra-red spectrum and also X-ray diffraction spectrum. Using the periodate oxidation procedure, the consumption of periodate per 1 mole of sialic acid was 5 moles and the formation of formaldehyde was 1 mole. It also contained 1 mole of the acetyl group but the glycol group was not detected

精製ニワトリ・オボムコイドの焦点電気泳動(農芸化学部門)

鶏卵白から精製されたオボムコイドを焦点電気泳動法を用いて分画した。オボムコイドは等電点3.52,3.85,4.00,4.15,4.35および4.50の6成分に分画されたが, 脱フラビン・オボムコイドではpH 3.52のピークは認められなかった。オボムコイドの生物活性の一つであるトリプシン阻害活性はpI 3.52,3.85,4.00および4.15の各画分に認められたが, pI 4.35および4.50のピークには認められなかった。また逆に, フラビン結合能はpI 4.15,4.35および4.50の画分に存在し, それ以外のピークには認められなかった。なお本実験に用いた焦点電気泳動の装置は自作したものであるが, 再現性のある良好な結果が得られた。The purified chicken ovomucoid was further fractionated with the method of isoelectrofocusing. The ovomucoid was fractionated into six fractions having the pI value of 3.52,3.82,4.00,4.15,4.35 and 4.50. The peak of protein with the pI value of 3.52 was not detected in the deflavo-ovomucoid. The trypsin inhibitory activity which was considered to be one of the biological activity of ovomucoid was detected in the fractions of pI value of 3.52,3.85,4.00 and 4.15,and the proteins of pI value of 4.35 and 4.50 did not show the trypsin inhibitory activity. Contrary to the results above mentioned, the flavin binding activity occurred in the peaks of pI value of 4.15,4.35 and 4.50,and it was not observed in the other peaks. The apparatus for the isoelectrofocusing used in these experiments was designed and prepared in our laboratory. The satisfactory and reproducible results were obtained with these equipments

Changes of Bovine κ-Casein Stored in Various Solutions (Agricultural Chemistry)

UHT滅菌乳の変質改良のための基礎データの蓄積, あるいは, 乳たん白質の保存特性の解明を目的として, 14種類の水溶液中に牛乳κ-カゼインを保存し, 保存によるκ-カゼインの諸性質の変化を検討した。即ち, 10% glucose水溶液, 10% lactose水溶液, 10% maltose水溶液, 10% sucrose水溶液, 2% NaCl水溶液, 2% glycine水溶液, 蒸留水, 10% glucose-4.5M urea, 10% lactose-4.5M urea, 10% maltose-4.5M urea, 10% sucrose-4.5M urea, 2% NaCl-4.5M urea, 2% glycine-4.5M urea, 4.5M ureaの各溶液に, κ-カゼインを0.25%(w/v)となるように溶解し, それらを5℃で保存した。κ-カゼインの保存状態を, α_-カゼイン安定化能, 4.5M尿素を含むディスクゲル電気泳動, セファアクリルS-300ゲルロ過クロマトグラフィー, フリーアミノ基およびスルフヒドリル基の定量によって検討した。α_-カゼイン安定化能は, 経時的に低下した。10% maltose水溶液中, 6ケ月保存では70.2%の安定化能を示したが, sucrose溶液中では沈殿が生じた。ゲル電気泳動では, κ-カゼインの分解と高分子化が同時進行していることが示唆された。一方, ゲルロ過クロマトグラフィーでは, 尿素を含まない保存液の場合は, ほとんど変化は観察されず, 尿素存在下保存の場合は, self-association能の低下が観察された。κ-カゼインの分解や高分子化は, フリーアミノ基およびスルフヒドリル基の定量から, 交互に進行し, アミノカルボニル反応やジスルフィド結合によるものと考えられた。Changes of bovine κ-casein stored in various solutions were followed by the stabilizing ability test for α_-casein in the presence of calcium ion, disc gel electrophoresis containing 4.5M urea, Sephacryl S-300 gel filtration chromatography, the determinations of free amino group and sulfhydryl group for the sake of accumulating the fundamental data for the improvement of denaturation of ultra high temperature sterilized milk and making clear the preservative characteristics of milk proteins. κ-Casein was stored at 5℃ as the solution dissolved in the following solutions; 10% glucose, 10% lactose, 10% maltose, 10% sucrose, 2% NaCl, 2% glycine, distilled water, 10% glucose-4.5M urea, 10% lactose-4.5M urea, 10% maltose-4.5M urea, 10% sucrose-4.5M urea, 2% NaCl-4.5M urea, 2% glycine-4.5M urea and 4.5M urea. The stabilizing ability was lost with the passage of time. κ-Casein stored in 10% maltose solution for 6 monthes had 70.2% of stabilizing ability, while 10% sucrose for 5 monthes began to precipitate. The results of electrophoreses indicated that the hydrolysis and polymerization of κ-casein progress at the same time. The lowering of self-asscoiation ability was observed by the gel chromatography of κ-casein stored in solutions containing urea. It was considered that the polymerization of κ-casein was due to the disulfide bond and the amino-carbonyl reaction

クロレラの経口投与の高血圧自然発症ラットにおよぼす影響(農芸化学部門)

高血圧自然発症ラット(SHR)を用いて, 26.4%のクロレラを含む食餌の尾動脈圧への影響を検討した。併せて, 体重変化, 臓器重量, 血清脂質含量をも測定した。対照群には, 市販固型飼料を与え, 実験期間は11日間であった。SHRは, クロレラ食で順調な生長を示し, 肝臓および小腸の重量も対照群とほぼ同じであった。盲腸重量は, 対照群の方が大きかったが, これは繊維含量が対照群の方が大きかったことによるものと考えられた。血清中の総コレステロール, 遊離コレステロール, HDL-コレステロールおよびトリグリセリド含量は, 両群とも正常であった。HDL-chol./Total-chol.比は, クロレラ食群の方が大であった。対照群の血圧は, 実験開始前に比べて上昇したが, クロレラ食群のそれは, 低下しており, クロレラ中に血圧降下作用を有する物質の存在を示した。Effect of chlorella diet (containing 26.4% of chlorella powder) on the blood pressure of spontaneously hypertensive rats (SHR) was examined. Further, weights of body, liver, small intestine and ceacum and concentrations of serum lipids of SHR were measured. The commercial diet was given to SHR as a control. Feeding period was 11 days. SHR fed chlorella diet grew adequately as expected. Weights of liver and small intestine of SHR fed chlorella diet were very similar to those of the control group. Ceacum of the control group was larger than that of chlorella diet group. This difference may be due to the fact that the commercial diet contained more fiber compared with the chlorella diet. The concentrations of total-, free-, HDL-cholesterol and triglyceride in serum of SHR were normal in any cases. The ratio of HDL-cholesterol to total-cholesterol of chlorella diet group was larger than that of control group. The blood pressure of control group rose compared with that before the experiment. On the contrary, the blood pressure of SHR fed chlorella diet lowered. These facts indicate the presence of anti-hypertensive material in chlorella