Studies on κ-Casein of Bovine Milk V : Chemical modification of κ-casein (Agricultural Chemistry)

κ-カゼインのアミノ基, カルボキシル基, SH基, チロシン, トリプトフアン, リジン, セリン, ヒスチジン, アルギニン, およびメチオニンを種々の方法で化学修飾し, それらがα_S-カゼイン安定化作用に及ぼす影響について調べた。今回の修飾反応条件であるpH7∿9においてはκ-カゼインはじゅうぶんに解離していないため, 反応基がκ-カゼイン複合体の表面に位置しているかどうかによって反応速度, ひいては修飾率が大きく左右された。アミノ基とカルボキシル基を修飾するとほぼ完全に安定化作用が消失し, ヒスチジンとチロシンの修飾も顕著な安定化力低下をもたらした。また, 還元してより低分子化すると未修飾κ-カゼインより安定化力が高まった。その他のアミノ酸残基の修飾はα_S-カゼインの安定化にほとんど無関係であった。焦点電気泳動による分析で, 6M尿素中においてκ-カゼインの等電点がpH5より酸性側へ移ると急にα_S-カゼインに対する安定化作用が失われることが判明した。デンプンゲル電気泳動によりκ-カゼインの化学修飾は分子電荷のみならず分子の大きさも変化させることが判明し, 特にセリン, ヒステジンそれにチロシンを修飾したものは会合が進み, 他方アミノ基とSH基を修飾したκ-カゼインはより小さい分子へ解離した。κ-Casein was chemically modified in various ways. Modified groups include; NH_2 group, COOH group, tyrosine, tryptophan, lysine, SH group, serine, histidine, arginine and methionine. These groups and residues were not completely modified, probably because κ-casein was not dissociated into single molecules under the conditions used. Normally κ-casein has an S_ of about 14,which decreases to about 3 when dispersed by alkali or urea. Modification of NH_2 and COOH groups resulted in almost complete loss of the stabilization ability. Modification of histidine and tyrosine fairly well promoted a decrease in this function. Reduced κ-casein stabilized interestingly more α_S-casein than native κ-casein did. Modification of other amino acids had little effect on the stabilization ability. Results of isoelectric focusing indicate that κ-casein was unable to maintain its stabilization function when its isoelectric point in 6M urea moved toward acidic side beyond pH5.0. Six components of reduced κ-casein were clearly separated by isoelectric focusing in 6M urea. We observed that components with isoelectric points at the neutral side were most susceptible to modification. These components seem to occupy the surface of the κ-casein complex. Chemical modification was shown to result not only in changes in molecular charge, but in changes in molecular size

Studies on κ-Casein of Bovine Milk III : Some properties of κ-casein and its complex (Agricultural Chemistry)

κ-カゼインを三種の方法で調製し, 電気泳動, 超遠心分析, α_S-カゼイン安定化力等について調べた。尿素中における等電点はpH6.0であり, α_S-カゼインとは違うことが明確になった。S_は尿素中で2.6∿3.8であり尿素が存在しないとpH8.0で14.4を示した。ヘキソースは1.5%, シアル酸は0.8%, リンは0.2%, チッ素は14%程度であった。DEAEセルロースクロマトグラフィーによって分画される前後でアミノ酸組成には変化がなかったが, α_S-カゼイン安定化力は分画後増大する傾向にあった。κ-Caseins were prepared by the calcium ethanol method, the Sephadex method and the urea sulfuric acid method. Some important properties of κ-caseins were investigated using isoelectric focusing, starch gel electrophoresis, ultracentrifugation, chemical analysis, stabilizing test of α_S-casein, and rennin treatment. Isoelectric focusing established that κ-casein had its isoelectric point near pH6.0 in 6M urea, usually accompanied by a second peak around pH5.6. Ultracentrifugation, however, showed a single peak having a S_ value of 2.6-3.8 in the presence of 6M urea and of 14.4 in the absence of such dispersing reagents. Normal contents of hexose, sialic acid, phosphorus, and nitrogen were respectively about 1.5,0.8,0.2,and 14%. Relative patterns of amino acid composition were similar in all the κ-caseins. In addition, amino acid composition in intact κ-casein and in the further purified κ-casein which formed the second peak in DEAE cellulose chromatography were almost identical, indicating that the κ-casein of the first peak is not an impurity but is one of the components which formed the original κ-casein complexes. The ability of κ-caseins to stabilize α_S-casein in the presence of calcium increased when purified by DEAE cellulose chromatography

Studies on egg protein VI : The trypsin inhibitor and flavomucoid fraction of ovomucoid (Agricultural Chemistry)

卵白から硫安分別, DEAE-C処理によってオボムコイドを純化し, CM-Cカラムクロマトグラフィーによりオボムコイドは4成分たんぱく(I∿IV)から組成されていることを明らかにした。オボムコイド4成分たんぱくのうち, I, IIにのみ抗トリプシン活性が認められ, ともにトリプシンと等モル作用して阻害することがわかり, またその活性はpH8.0において100°, 20分;90°, 30分;80°, 170分の加熱処理で完全に失活した。IIIおよびIV成分たんぱくは, フラボムコイドK_1,K_2のアポたんぱくであり, いずれもFRと等モルで結合し, その結合割合はpH7.0付近が最も高く, また結合能は80°以上の加熱では55分以内で, 70°加熱では2時間でなくなった。The ovomucoid was purified by the DEAE-C treatment after the ammonium sulfate fractionation of egg white. It is demonstrated by the CM-C column chromatography that the ovomucoid consisted of four protein components (I-IV). The antitryptic activity was present only in components I and II, and both components inhibit trypsin by forming the equimolar complexes. The antitryptic activity was inactivated completely by the heating at 100℃ for 20 minutes, at 90℃ for 30 minutes and 80℃ for 170 minutes at pH8.0. The components III and IV are the apoprotein of falvomucoid K_1 and K_2; both components bind flavin to form the equimolar complex. The optimum pH for FR-binding was 7.0 and the flavin-binding capacity was inactivated by heating for 55 minutes at 80℃ and also 2 hours of heating at 70℃

Studies on κ-Casein of Bovine Milk I : Purity of κ-casein prepared by several different methods (Agricultural Chemistry)

五種類のκ-カゼイン調製法を比較検討した結果, 収量の点では各方法とも酸カゼイン43gより2ないし3gで有意の差は認められなかった。一方, セファデックスゲルろ過とデンプンゲル電気泳動によって純度を比較したところMckejieらのカルシューム・エタノール法とYaguchiらのセファデックス法が好結果を与えた。しかし, 純度の点で問題のあったSwaisgoodらのTCA法, Zittleのの尿素硫酸法でも, 各々セファデックス処理を加えること, 酢酸アンモンによる沈殿形成時の温度条件を厳守することにより高純度のκ-カゼインを調製しうることを認めた。A comparison of five procedures for preparing κ-casein from the acid casein of bovine milk was made based on the criterion of purity using Sephadex gel filtration and starch gel electrophoresis. There were no significant differences in yields among the five procedures, by which two to three grams of κ-casein were obtained from 43 grams of acid casein. But the κ-casein prepared using the calcium ethanol method described by Mckenjie and Wake or by the Sephadex method described by Yaguchi et al. was very pure. It was also shown that it is possible to prepare reasonably pure κ-casein using the trichloroacetic acid method, described by Swaisgood and Brunner, combined with Sephadex treatment, and using the urea sulfuric acid method described by Zittle and Custer when temperature was strictly controlled in the precipitation step of κ-casein by ammonium acetate

Studies on κ-Casein of Bovine Milk IV : The binding of calcium to α_S-, and κ-casein (Agricultural Chemistry)

Caとカゼインとの反応をCa濃度0∿10mMの範囲で調べた。3mM濃度で沈殿し始めるα_S-カゼインはκ-カゼインによって完全に安定化された。一方, κ-カゼイン自身もCaの存在下で会合を起こし, 多分散状態が著るしくなったが, 沈殿はしなかった。Caが存在しない場合, κ-カゼインとα_S-カゼインのS_は各々14.4と1.8であったが, Ca濃度の増加と共に会合していく様子が超遠心分析とゲルろ過によって確認された。Caの存在下でα_S-カゼインは時間と共に会合度を増し, ついには沈殿するがα_S-κ-カゼイン複合体や, κ-カゼイン単独の場合は時間的変化が極めて緩慢であった。pH7と8におけるカゼインによるCaの吸着量を, Ca濃度1∿10mMの範囲で調べた。α_S-カゼインの沈殿に必要な吸着Ca量はカゼイン1モル当り約12モルであった。一般的にいって, Caとの反応でα_S-κ-カゼイン複合体のCa吸着能はα_S-, κ-カゼイン単独の場合の吸着能から推定されるより強かった。一方, κ-カゼインのCa吸着能は著るしく弱いことが明白となった。その他吸着の時間的変化についても調べた。Reactions between Ca and casein were investigated at various Ca concentrations ranging from 0 to 10mM. α_S-Casein began to precipitate at 3mM and was completely stabilized by κ-Casein tended to aggregate randomly in the presence of 5mM Ca. S_ values were 14.4 and 1.8 for κ-casein and α_S-casein, respectively, in the absence of Ca. Caseins were found in gel filtration to be brought into polymerization at such low Ca concentrations that no aggregated particles were visible. α_S-κ Complex did not grow as fast as α_S-casein did in the presence of Ca. The amounts of Ca bound to caseins were determined at pH7 and 8 in the presence of 1 to 10mM Ca using; equilibrium dialysis, gel filtration and centrifugation. The minimum amount of Ca necessary for the initiation of α_S-casein precipitation is posited to be about 12 moles per mole casein. Weak adsorption of Ca, due to some structural factor, was indicated since the amounts of bound Ca obtained by centrifugation were remarkably lower than those determined by the other two methods. The state of bound Ca were studied by gel filtration of ^Ca-caseinates with an eluant containing no Ca. More Ca was bound to the α_S-κ complex in the early stage of the reaction than to the same amount of α_S-casein only. But the amount of Ca bound to the complex did not vary with reaction time, while that bound to α_S-casein increased greatly, accompanied by the progress of polymerization. The intensity of Ca binding to κ-casein was so weak that only a small amount of Ca was eluted together with κ-casein in the gel filtration of Ca-κ-caseinate

Studies on egg protein VI : The trypsin inhibitor and flavomucoid fraction of ovomucoid (Agricultural Chemistry)

卵白から硫安分別, DEAE-C処理によってオボムコイドを純化し, CM-Cカラムクロマトグラフィーによりオボムコイドは4成分たんぱく(I∿IV)から組成されていることを明らかにした。オボムコイド4成分たんぱくのうち, I, IIにのみ抗トリプシン活性が認められ, ともにトリプシンと等モル作用して阻害することがわかり, またその活性はpH8.0において100°, 20分;90°, 30分;80°, 170分の加熱処理で完全に失活した。IIIおよびIV成分たんぱくは, フラボムコイドK_1,K_2のアポたんぱくであり, いずれもFRと等モルで結合し, その結合割合はpH7.0付近が最も高く, また結合能は80°以上の加熱では55分以内で, 70°加熱では2時間でなくなった。The ovomucoid was purified by the DEAE-C treatment after the ammonium sulfate fractionation of egg white. It is demonstrated by the CM-C column chromatography that the ovomucoid consisted of four protein components (I-IV). The antitryptic activity was present only in components I and II, and both components inhibit trypsin by forming the equimolar complexes. The antitryptic activity was inactivated completely by the heating at 100℃ for 20 minutes, at 90℃ for 30 minutes and 80℃ for 170 minutes at pH8.0. The components III and IV are the apoprotein of falvomucoid K_1 and K_2; both components bind flavin to form the equimolar complex. The optimum pH for FR-binding was 7.0 and the flavin-binding capacity was inactivated by heating for 55 minutes at 80℃ and also 2 hours of heating at 70℃

Studies on κ-Casein of Bovine Milk VI : Reduced components and the α_S-casein stabilizing ability of succinylated κ-casein (Agricultural Chemistry)

Succinic anhydrideによってκ-カゼインのアミノ基を4.8∿93.2%修飾したサンプルを用いて, α_S-カゼイン安定化力, 構成成分の被修飾性, 等電点変化, 会合度変化, Caとの反応性を調べた。α_S-カゼイン安定化力は, 8.8%修飾までは変化なく, 15.9∿19.6%修飾で次第に低下し, 32.3%修飾で完全に消失した。このように最も反応性に富んだアミノ基がα_S-カゼイン安定化力に無関係である事実は, κ-カゼインのアミノ基は直接α_S-カゼインとのミセル形成に関係していないことを意味するものと考えられる。還元κ-カゼインの電気泳動の結果, κ-カゼイン構成成分のうち尿素中でpH6付近に等電点を持つ成分がpH5付近の成分より, より早く修飾されることが判明した。したがって, より中性側に等電点を持つ成分がκ-カゼインの表面構造を形成しているものと推定された。サクシニル化に伴なって等電点が酸性側へ移行し, 尿素中における等電点がα_S-カゼインのそれと同じpH5付近になると, Caとの反応性が低下し, α_S-カゼインを安定化する能力が完全に失なわれた。Amino groups of κ-casein were succinylated at pH8 to various degrees ranging from 4.8 to 93.2 percent. These modified κ-caseins were used for α_S-casein stabilizing test in the presence of 0.02M calcium chloride. The ability of κ-casein to stabilize α_S-casein did not change up to 8.8 percent succinylation, but decreased gradually as the modification reaction proceeded further until 32.3 percent succinylation, where the α_S-casein stabilizing ability of κ-casein completely disappeared. The initial succinylation of amino groups occurred on specific residues rather than in a random fashion among all the free residues. At pH8,the κ-casein components with isoelectric points at pH5.8 and 6.1 in 6M urea were, in the early stage of succinylation, modified more rapidly than the rest with isoelectric points on more acidic side. It indicates that the κ-casein components with their pIs around pH6 are located in the external hydrophilic region of κ-casein complex, and that they probably play important roles in the interaction with α_S-casein which brings about the stabilization of α_S-casein in the presence of calcium chloride. As previously reported, κ-casein lost its ability to stabilize α_S-casein when the isoelectric point moved to pH5.0-5.2,that of α_S-casein, in 6M urea as a result of succinylation

Biochemical studies on chick embryo during incubation (Agricultural Chemistry)

孵卵中の卵白のシアル酸およびトリプシン阻害活性の変化を追求した。シアル酸濃度は日と共に徐々に増加し, インヒビター活性は, 最初減少したのち増加し, それ以後は変化しなかった。孵卵数日にして形成される漿尿膜(CAM)は, 強いシアリダーゼ活性を有し, 卵白のシアロタンパク質であるオボムチンやオボムコイドからシアル酸を遊離させた。またCAM自身もシアル酸を有し, 自らもシアル酸を遊離させる。微生物由来のシアリダーゼで処理したCAMは非常に強いシアリダーゼ活性を示したが, これは加えたシアリダーゼが膜と強く結合するものと考えられる。シアロタンパク質とCAMの結合が, オボムコイドの示すトリプシン阻害活性をもって間接的に, ^Iによる標識で直接的に検討された。用いたオボムコイド, オボムチン共に脱シアル酸処理をしたCAMとよく結合し, 逆に脱シアル酸処理したタンパク質は, そのままのCAMとよく結合した。The change of sialic acid content and trypsin inhibitory activity of chicken egg white during incubation were studied. The sialic acid concentration of egg white slightly increased during incubation, indicating the absorption of sialo-proteins of egg white into embryo occurred later than that of others. In early period of incubation, trypsin inhibitory activity rapidly and extensively decreased and after this period, trypsin inhibitory activity was kept at approximately constant level. Neuraminidase (sialidase) of chorioallantoic membrane (CAM) was able to release the endogenous sialic acid and showed the strong activity toward exogenously added ovomucoid and ovomucin. When neuraminidase was mixed with CAM in a buffer, sialic acid at the membrane surface was removed. Added neuraminidase remained bound at the surface of CAM despite of sufficient washing and shows strong activity on sialo-proteins. The experiments for CAM-sialo-protein binding showed that desialylated proteins (desialylated ovomucoid and ovomucin) bound to native CAM in greater amount than to desialylated CAM. Further, it was found that native sialo-proteins (native ovomucoid and ovomucin) bound to desialylated CAM rather than native CAM. ^I-labelled sialo-proteins were prepared and used in these binding experiments

Optical studies on the purified eggplant trypsin inhibitor (Agricultural Chemistry)

ナス果皮より均一に精製したトリプシン・インヒビターについてその光学的性質を検討した。各種pHにおいて, 紫外吸収スペクトルを測定したがpH 3.1から9.4の範囲では, チロシンおよびフェニールアラニン残基による同じスペルトルを示したが, pH 10以上ではチロシン残基のイオン化による著しい変化が認められた。またアミノ酸分析で確認されているようにトリプトフアンを含まないことは, 紫外吸収スペルトルからも明らかであった。275nmで励起した蛍光スペクトルが測定されたが, pH 3.1から9.4の範囲では蛍光強度は, ほゞ一定であり10以上で減少した。またCDスペクトルの測定結果はこのインヒビターが, ほとんどヘリックス構造やβ-構造を含まないことを示している

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