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ホームトリプシンインヒビター

トリプシンインヒビター

天然トリプシンインヒビターはセリンプロテアーゼ阻害剤(セルピン)としても知られ、プロテアーゼ阻害剤の中で最も種類が多い最大のファミリーです。1セルピンはin vivoでセリンプロテアーゼを阻害することでタンパク質の活性および異化をコントロールします。2

トリプシンインヒビターは、ウシ膵臓、オボムコイド、大豆、ライマメの4種類の天然由来のものがあります。各阻害剤は基質の競合的アナログとして作用し、セリンプロテアーゼに結合して不活性複合体を形成し、プロテアーゼを不活化します。3

This process allows the serpin (trypsin inhibitor) to stop the proteolytic activity of the serine protease when its function is no longer necessary.

Trypsin inhibitors provide unique processes depending on their source. For example, inhibitors in the seeds of legumes (soybean and lima bean) act as a feeding deterrent for insects by disrupting midgut proteases. This natural function is being expanded upon in the development of insect resistant transgenic plants. Soybean inhibitors have also been found to contribute to pancreatic hypertrophy in rats, again providing a feeding deterrent. The Bowman-Birk soybean inhibitor is being studied as a cancer preventive agent.4

Products

トリプシンインヒビター(ウシ膵臓)

図1.T0256-ウシ膵臓由来トリプシンインヒビター

別名:BPTI(ベーシック膵臓トリプシンインヒビター)
分子量:~6.5 kDa(単鎖58アミノ酸ペプチド)
pI:~10.5
比活性:トリプシンインヒビター1 mgは、タンパク質1 mg当たり約10,000 BAEE unitsの活性を有するトリプシン1.5 mg超を阻害します
ニュージーランド産ウシ膵臓に由来
溶解性:本製品は2 mg/mLで水に溶けます

BPTI is a 58 amino acid single polypeptide chain with 3 disulfide bonds.BPTIはアミノ酸58個から成るポリペプチド単鎖で、3つのジスルフィド結合を有します。5BPTI inhibits both bovine and human trypsin, chymotrypsin, kallikrein and plasmin. BPTI does not inhibit porcine elastase.6


トリプシンインヒビター(ニワトリ卵白由来)

図2.T9253-ニワトリ卵白由来トリプシンインヒビター(II-O型)

別名:オボムコイド
分子量:~28 kDa
pI:4.17
比活性:1 mgは、タンパク質1 mg当たり約10,000 BAEE unitsの活性を有するトリプシン0.8~1.2 mgを阻害します。また、タンパク質1 mg当たり約40 BTEE unitsの活性を持つキモトリプシン0.3 mg以下を阻害する場合があります。
溶解性:本製品は0.67 Mリン酸緩衝液(pH 7.6、2 mg/mL)に溶けます。

Chicken ovomucoid is a major glycoprotein that inhibits bovine trypsin. It is comprised of 186 amino acids that are arranged in three tandem domains.ニワトリオボムコイドはウシトリプシンを阻害する主要糖タンパク質の1つです。186個のアミノ酸から成り、縦に並んだ3つのドメインを形作っています。8各ドメインには3つのジスルフィド結合、2つのチロシン残基、1つの活性部位があります。9オボムコイドは各オボムコイド分子がトリプシンと1:1で結合する、ヘッド領域が1つのトリプシンインヒビターです。7

This product contains some ovoinhibitor. Ovoinhibitor is another protease inhibitor from chicken egg white. Ovoinhibitor has at least five binding sites, and is responsible for the inhibition of bovine trypsin, bovine chymotrypsin and porcine elastase.本製品はオボインヒビターを若干含んでいます。オボインヒビターは、オボムコイドとは別のニワトリ卵白由来プロテアーゼ阻害剤です。オボインヒビターには5つ以上の結合部位があり、ウシトリプシン、ウシキモトリプシン、およびブタエステラーゼを阻害します。10トリプシンおよびキモトリプシンの結合部位はそれぞれ2か所あり、残り1つの部位にはエラスターゼが結合します。11オボムコイドおよびオボインヒビターはニワトリ卵白中に豊富に存在するプロテアーゼ阻害物質で、ニワトリニワトリ卵白タンパク質にそれぞれ11%および1.5%含まれています。12

T2011-ニワトリ卵白由来トリプシンインヒビター

Type III-O(オボインヒビターフリー)
別名:オボムコイド
分子量:~27 kDa
比活性:1 mgは、タンパク質1 mg当たり約10,000 BAEE unitsの活性を有するトリプシン1.0~2.0 mgを阻害します。
溶解性:本製品は0.67 Mリン酸緩衝液(pH 7.6、2 mg/mL)に溶けます。

This product has the same characteristics as described above for product T9253, but is further purified by an ammonium sulfate cut and filtration process to eliminate the ovoinhibitor. The absence of ovoinhibitor results in an even more pure ovomucoid that continues to inhibit trypsin in a 1:1 complex.本製品は上述の製品T9253と同じ特徴を持っていますが、硫酸アンモニウムおよびろ過プロセスによりさらに精製し、オボインヒビターを除去しています。オボインヒビターを含まないため、オボムコイドの純度が高くなり、トリプシンと1:1の比で複合体を形成し、持続的に阻害します。

T4385 - Trypsin inhibitor from turkey egg white

Type II-T
Synonym: Turkey Ovomucoid
M.W.: ~20 kDa
Specific Activity: One mg will inhibit 0.9-1.3 mg of trypsin with activity of ~10,000 BAEE units per mg protein or 0.4-1.0 mg of α-chymotrypsin with activity of ~40 BTEE units per mg protein
Solubility: This product is soluble in 0.67 M phosphate buffer, pH 7.6 (1 mg/mL)

Trypsin inhibitor from turkey egg white contains two independent binding sites, one for bovine trypsin and the other for α-chymotrypsin. At low ph (2.0) a third domain (OMTKYT3) develops and inhibits most serine proteases that prefer a neutral complex site.13

七面鳥卵白由来トリプシンインヒビターには独立した2つの結合部位があり、1つはウシトリプシン、もう1つはα-キモトリプシンに結合します。低pH値(2.0)では3つ目のドメイン(OMTKYT3)が出現し、中性錯体に親和性がある大半のセリンプロテアーゼを阻害します。13

KunitzおよびBowman-Birk(BBI)大豆プロテアーゼインヒビター

These two proteins are the most abundant protease inhibitors in soybeans. BBI with a molecular mass of 8 kDa is a strong inhibitor of both trypsin and chymotrypsin and contains independent binding sites for each. The Kunitz inhibitor with a molecular mass of 20.1 kDa, comprises one binding site that strongly inhibits trypsin while weakly binding chymotrypsin.

トリプシンインヒビター(Kunitz)(大豆)

図3.Glycine max(大豆)由来トリプシンインヒビター:Kunitzインヒビター

別名:Kunitzトリプシンインヒビター、Tia1、STI、およびSBTI
分子量:20.1 kDa
pI:4.525
吸光係数:E1% = 9.94
(280 nm、pH 7.6緩衝液)

Soybean trypsin inhibitor was first isolated by Kunitz.大豆トリプシンインヒビターはKunitzによって初めて単離されました。14他にも数種の関連インヒビターが大豆から発見されています。15大豆由来トリプシンインヒビターは単量体タンパク質であり、181個のアミノ酸残基から成るポリペプチド単鎖に2つのジスルフィド架橋があります。16-18

Soybean trypsin inhibitor inhibits trypsin, and to a lesser extent chymotrypsin大豆トリプシンインヒビターはトリプシンを阻害し、キモトリプシン19およびプラスミンの阻害はトリプシンに対する阻害よりも弱いです。20大豆トリプシンインヒビターはトリプシンに対する阻害と同じ機構により、トリプシン以外のプロテアーゼも阻害します。SBTIは血漿カリクレインおよび凝固因子Xaに対する阻害作用も有しています。しかし、大豆トリプシンインヒビターはメタロプロテアーゼ、組織中カリクレイン、酸性プロテアーゼ、およびチオプロテアーゼを阻害しません。Soybean trypsin inhibitor forms a 1:1 stoichiometric complex with the protease active site. Upon formation of this complex, trypsin may cleave a single arginine-isoleucine bond on the inhibitor.21,22 Inhibition is both reversible and pH dependent. Dissociation of this complex may yield a modified or native form of the inhibitor.23 The optimal pH for trypsin binding is 8.0 with an association constant of greater than 109 at pH 8.0 and an association constant 0.15-2.6 x 104 at pH 3.6-4.4.24


T9003 - Glycine max(大豆)由来トリプシンインヒビター
Type I-S
本品はDEAEセファロースを用いたクロマトグラフィーで精製しており、10%リン酸ナトリウム緩衝塩(pH 7.6)を含みます。
比活性:1 mgは、タンパク質1 mgあたり約10,000 BAEE unitsの活性を有する1.0~3.0mgのトリプシンを阻害します。
キモトリプシン1 unitはpH 7.8、25℃で1分間に1.0 µmolのBTEEを加水分解します。
溶解性:トリプシンインヒビターは水およびリン酸バッファーに溶けます(10 mg/mL)。平衡塩類溶液(1 mg/mL)および血清フリー培地に溶けます。濃度10 mg/mLを超える溶液は濁った黄色~琥珀色を呈する場合があります。
保管/安定性:分注凍結後、本製品は−20℃で活性を保ちますが、凍結解凍は避けてください。このタンパク質を80℃まで短時間加熱すると可逆的な変性を起こし、90℃にまで短時間加熱すると不可逆的な変性を起こします。15

T6522 - Trypsin inhibitor from Glycine max (soybean)
Type I-S
Cell Culture tested
This product is chromatographically purified on DEAE Sepharose and contains 10% Sodium phosphate buffer salts, pH 7.6.
Specific Activity: One mg will inhibit 1-3 mg of trypsin with activity of ~10,000 BAEE units per mg protein
Solubility: Trypsin inhibitor is soluble in water and phosphate buffers at 10 mg/mL. It is soluble in balanced salt solutions (1 mg/mL) and in serum-free media. Solutions at concentrations higher than 10 mg/mL may be hazy and have a yellow to amber color.
Storage/Stability: This product remains active in frozen aliquots at –20 °C, but freeze-thaw should be avoided. This protein is reversibly denatured by short heating to 80 °C and irreversibly denatured by heating to 90 °C.15


T6414 - Trypsin inhibitor from Glycine max (soybean)
This product is a sterile filtered, cell cultured 1x solution (0.1% trypsin inhibitor solution in Dulbecco's PBS), which is appropriate for use in cell culture applications. It has been optimized for passage of endothelial cells.
Storage/Stability: This product remains active in frozen aliquots at –20 °C, but freeze-thaw should be avoided. This protein is reversibly denatured by short heating to 80 °C and irreversibly denatured by heating to 90 °C.15


T9008 - Trypsin inhibitor from Glycine max (soybean) 1% solution in water
For use in cell culture applications
Prepared from T9003, sterile filtered and processed to yield a convenient no buffer solution.
Storage/Stability: This product is stored at 2-8 °C. This solution can also be stored as frozen aliquots at –20 °C, but freeze-thaw cycles should be avoided. This protein is reversibly denatured by short heating to 80 °C and irreversibly denatured by heating to 90 °C.15

T9008-Glycine max(大豆)由来トリプシンインヒビター1%水溶液
細胞培養用
T9003調製液を滅菌ろ過し処理した緩衝液を含まない便利な溶液です。
保管/安定性:本製品は2~8℃で保存します。本製品は分注凍結し−20℃で保存できますが、凍結解凍は避けてください。このタンパク質を80℃まで短時間加熱すると可逆的な変性を起こし、90℃にまで短時間加熱すると不可逆的な変性を起こします。15

T9128 - Glycine max(大豆)由来トリプシンインヒビター
Type II-S
硫酸アンモニウム分画で調製し、一連の精製ステップを経て、組成90%タンパク質-10%リン酸ナトリウム緩衝塩(pH 7.6)にした製品
比活性:1 mgは、タンパク質1 mg当たり約10,000 BAEE unitsの活性を有するトリプシン1.0 mg以上を阻害します。
溶解性:本製品は水に溶けます(1 mg/mL)。濃度10 mg/mLを超える溶液は濁った黄色~琥珀色を呈する場合があります。
保管/安定性:分注凍結後、本製品は−20℃で活性を保ちますが、凍結解凍は避けてください。このタンパク質を80℃まで短時間加熱すると可逆的な変性を起こし、90℃にまで短時間加熱すると不可逆的な変性を起こします。15

T2327 - Trypsin inhibitor from Glycine Max (soybean)
≥98% Kunitz type inhibitor
Further purified chromatographically from T9128 to yield pure Kunitz Type trypsin inhibitor.
This product contains 10% sodium phosphate buffer salts, pH 7.6.
Specific Activity: One mg will inhibit ≥ 1.6 mg of trypsin with activity of ~10,000 BAEE units per mg protein.
Solubility: Trypsin inhibitor is soluble in water and phosphate buffers at concentrations higher than 10 mg/mL may be hazy and have a yellow to amber color.
Storage/Stability: This product remains active in frozen aliquots at -20 °C, but freeze-thaw should be avoided. This protein is reversibly denatured by short heating to 80 °C and irreversibly denatured by heating to 90 °C.15


Trypsin-chymotrypsin inhibitor from Glycine max (soybean): Bowman-Birk inhibitor

Glycine max(大豆)由来トリプシン-キモトリプシンインヒビター:Bowman–Birkインヒビター
トリプシンインヒビター(Bowman-Birk)(大豆)

図4.T9777-Glycine max(大豆)由良トリプシン-キモトリプシンインヒビター

別名:Bowman–Birkインヒビター(BBI)
CAS 番号:37330-34-0
ユニット定義:トリプシン1 unit(反応液量3.2 mL)を、pH 7.6、25℃で基質BAEEと反応させると1分当たりのΔA253が0.001になります(反応液量3.2 mL、光路1 cm)。
分子量:8 kDa29

The Bowman-Birk inhibitor (BBI) from soybean is a monomeric protein containing 71 amino acids in a single polypeptide chain crosslinked by seven disulfide bridges.26 This inhibitor contains two independent inhibitory binding sites, one for trypsin and the other for chymotrypsin.27 BBI binds each protease to form a 1:1 complex. Since the inhibition is non-competitive, BBI has the ability to form a ternary complex with both enzymes.28

This product is chromatographically purified on DEAE Sepharose and contains 20% Sodium phosphate buffer salts, pH 7.6.
Specific Activity: One mg protein will inhibit ≥0.5 mg trypsin with activity of ~10,000 BAEE units per mg protein.
One mg protein will inhibit ≥1.0 mg chymotrypsin with activity of ~40 BTEE units per mg protein.
Solubility: Trypsin-chymotrypsin inhibitor is soluble in water or 0.67 M Sodium phosphate, pH 7.6 (1 mg/mL).

本製品はDEAEセファロースを用いたクロマトグラフィーで精製しており、20%リン酸ナトリウム緩衝塩(pH 7.6)を含みます。
比活性:1 mgはタンパク質1 mg当たり約10,000 BAEE unitsの活性を有するトリプシン0.5 mg以上を阻害します。
また1 mgは、タンパク質1 mg当たり約40 BAEE unitsの活性を有するキモトリプシン1.0 mg以上を阻害します。
溶解性:トリプシン-キモトリプシンインヒビターは、水および0.67 Mリン酸緩衝液(pH 7.6、1 mg/mL)に溶けます。

Phaseolus limensis(リママメ)由来トリプシンインヒビター:

Synonyms: Trypsin Inhibitor from lima beans and LBTI
M.W.: 9 kDa
pI: 4.525
Extinction Coefficient: E1% = 9.94
(280nm, pH 7.6 buffer)

Trypsin inhibitor from lima bean is a monomer comprised of 83 amino acids that has the ability to undergo a concentration-dependent dimerization. The degree of self-association depends upon the type of variant and pH.30

Trypsin inhibitor from Phaseolus limensis has four to six variants whose inhibitory activities towards trypsin are essentially identical; whereas, some differences exist towards inhibition of chymotrypsin. The variant’s amino acid sequences are similar, each containing 7 disulfide bonds.31

Trypsin inhibitor forms a 1:1 stoichiometric complex with the protease active site. The complex of the inhibitor with either trypsin or chymotrypsin has no further inhibitory effect toward more of the same enzyme, but has full activity towards the other enzyme (forming 1:1:1 complex).30,32 This implies that there is one binding site for trypsin and another for chymotrypsin. Trypsin binds to a Lys-Ser site, while chymotrypsin binds to a Leu-Ser site.31 Inhibition is both reversible and pH dependent. Dissociation of this complex may yield a modified or native form of the inhibitor.23 The optimal pH for trypsin binding is 8.0 with an association constant of greater than 109 at pH 8.0, and an association constant of 0.15-2.6 x 104 at pH 3.6-4.4.24

トリプシンインヒビターはプロテアーゼ活性部位と化学量論比1:1で複合体を形成します。複合体になったトリプシンインヒビターは、結合相手と同一酵素をさらに阻害することはなくなりますが、結合相手ではないトリプシンまたはキモトリプシンに対しては100%の阻害活性があります(1:1:1の複合体になる)。30,32これはトリプシン用とキモトリプシン用の結合部位が1つずつあることを意味します。トリプシンはLys-Ser部位に結合するのに対し、キモトリプシンはLeu-Ser部位に結合します。31この阻害は可逆的であると同時にpH依存性です。この複合体の解離により、元のインヒビターまたは修飾されたインヒビターが生じます。23トリプシン結合の至適pHは8.0で、pH 8.0での結合定数は109超、pH 3.6~4.4の結合定数は0.15~2.6 × 104 です。24

T9378 Phaseolus limensis(リママメ)由来トリプシンインヒビター
本製品は、10%リン酸ナトリウム緩衝塩(pH 7.6)を含む凍結乾燥粉末です。
比活性:1 mgは、タンパク質1 mg当たり約10,000 BAEE unitsの活性を有するトリプシン0.8 mg以上を阻害します。
溶解性:トリプシンインヒビターは水およびリン酸緩衝液に溶けます(1 mg/mL)。平衡塩類溶液および血清フリー培地に溶けます。濃度10 mg/mLを超える溶液は濁った黄色~琥珀色を呈する場合があります。


関連製品
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細胞培養での用途

Trypsin inhibitors are used in cell culture applications to further inhibit tryptic activity during cell dissociation to prevent cell damage/death.

Procedure:
After trypsinizing cells, resuspend cells in 1 mL trypsin inhibitor solution (1 mg/mL using either a balanced salt solution or serum free media) for every mL of trypsin solution used for dissociation. Centrifuge the cell suspension at 1000 rpm for 5 minutes. A cell pellet should form. Remove as much of the trypsin inhibitor as possible and resuspend the pellet in serum-free medium. Culture cells as desired.

トリプシンインヒビター活性のアッセイ法

The activity of most trypsin inhibitor preparations is determined by a continuous rate spectrophotometric assay and expressed as the inhibition of BAEE units.

Unit Definition: One BAEE unit will produce a ΔA253 of 0.001 per min at pH 7.6 and 25 °C using BAEE as a substrate. Reaction volume = 3.2 mL.

Conditions
Temp = 25 °C, pH = 7.6, A= 253 nm, Light path = 1 cm
In a 3.2 mL reaction mix, the final concentrations are 63 mM sodium phosphate, 0.23 mM Nα-benzoyl-L-arginine ethyl ester (BAEE), 0.002 mM hydrochloric acid, 0.005mg trypsin, and 0.003 - 0.001 mg trypsin inhibitor.

Reagents Needed
S0751 - Sodium Phosphate monobasic
B4500 - Nα-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE)
258148 - Hydrochloric acid ACS reagent
T8003 - Trypsin from bovine pancreas

Reagents

  1. 67 mM Sodium Phosphate Buffer, pH 7.6 at 25 °C
    (Prepare 100 mL in deionized water using Sodium Phosphate, Monobasic, Anhydrous, Product No. S0751. Adjust to pH 7.6 at 25 °C with 1 M NaOH.)
  2. 0.25 mM Nα-Benzoyl-L-Arginine Ethyl Ester Solution (BAEE)
    (Prepare 50 mL in Reagent a using Nα-Benzoyl-L-Arginine Ethyl Ester, Hydrochloride, Product No. B4500.)
  3. 1 mM Hydrochloric Acid Solution (HCl)
    (Prepare 50 mL in deionized water using concentrated Hydrochloric Acid, Product No. 258148.)
  4. Trypsin Enzyme Solution (Trypsin)
    (Immediately before use, prepare a solution containing 1 mg protein/mL of Trypsin, Product No. T8003, in cold Reagent C.)
  5. Trypsin Inhibitor Solution (Inhib.)
    (Immediately before use, prepare a solution containing 1.0 mg/mL of Trypsin Inhibitor in cold Reagent A.)

Procedure
Pipette (in milliliters) the following reagents into suitable quartz cuvettes

Part A:

25℃で5~6分間放置する。

以下の試薬を転倒混和し、ピペットで適切なキュベットに移します(mL):

Part B:

転倒混和し25 ℃になるまで平衡化します。適切な恒温分光光度計を用いて示度が安定するまでA253nmを測定します。次に下記を加えます:

直ちに転倒混和し、A253nmの増加分を約5分間記録する。試験試料、ブランク、およびインヒビター無添加溶液について、最大線形速度を用いてΔA253nm/分を算出します。

計算式

トリプシン活性:BAEE unit/酵素mL=

(ΔA253nm/min Test – ΔA253nm/min Blank)(df)(10.0)


(0.001)(0.10)(0.5)

df=希釈率
0.001=トリプシン1 unitあたりのA253nm/分(pH 7.6、25°C、反応液3.2 mL)
0.10=酵素使用量(mL)(パートB)
10.0=アッセイ総量(mL)(パートA)
0.5=酵素使用量(mL)(パートA)

Units/mg solid(固体)=


units/mL enzyme


mg solid/mL enzyme

トリプシン活性(BAEE unit/タンパク質mg)対トリプシンインヒビターmL/RM
トリプシンインヒビターmg=(トリプシンインヒビターmL)(トリプシンインヒビター濃度mg/mL)


トリプシンインヒビター1 mgで阻害されるトリプシンmg=

mg Trypsin/RM (normalizing factor)

mg Trypsin Inhibitor (from plot)

正規化係数=(非阻害トリプシンBAEE unit/mg solid(固体) / 標準トリプシン10,000 BAEE units)

注記:

  1. This enzyme assay is used to assay product numbers: T9003, T9008, T9128, T9253, T2011, T4385, T9378, and T0256.
  2. When assaying Trypsin Inhibitor, Type II-S, product number T9128, prepare a solution containing 0.60 mg/ml of Trypsin Inhibitor in cold Reagent a.
  3. The uninhibited Trypsin activity should be within 85% of the release value for activity.
  4. With 11,700 to 13,005 Trypsin units/mg solid per label, the acceptable range for activity of the uninhibited Trypsin reaction should be 10,000 to 15,300 Trypsin units/mg solid. This range should also correspond to a corrected ΔAbs253nm/minute of 0.0545 to 0.0835. With this rate and an inhibition of 20% to 80% the ΔAbs253nm/minute should be above the spectrophotometer rate detection limit of 0.0020.

Trypsin Unit Conversions
1 BAEE µM Unit = 200 BAEE Units
1 TAME µM Unit = 0.27 BAEE µM Units
1 BAEE µM Unit = 3.64 TAME Units
1 TAME µM Unit = 55 BAEE A253 Units
1 BAEE A253 Unit = 0.018 TAME µM Unit
1 TAME µM Unit = 180 TAME A247 Units
1 TAME A247 Unit = 0.33 BAEE Units
1 USP Unit = 3.0 BAEE Units
1 NF Unit = 1.1 USP Units

トリプシンunit換算
1 BAEE µM unit = 200 BAEE Units
1 TAME µM unit = 0.27 BAEE µM units
1 BAEE µM unit = 3.64 TAME units
1 TAME µM unit = 55 BAEE A253 units
1 BAEE A253 unit = 0.018 TAME µM units
1 TAME µM unit = 180 TAME A247 units
1 TAME A247 unit = 0.33 BAEE units
1 USP unit = 3.0 BAEE units
1 NF unit = 1.1 USP units

参考文献

1.
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