Trypsin Inhibitors

C.A.S.: 9035-81-1

Two bovine pancreatic trypsin inhibitors have been isolated. Both are produced by the acinar cells and provide security against accidental trypsinogen activation and consequential unbridled proteolysis. The secretory trypsin inhibitor isolated by Kazal et al. (1948) is secreted with the zymogens into the pancreatic juice. It has been reported on by Sealock and Laskowski (1973), Schweitz et al. 1973), Greene and Giordano (1969), Cerwinsky et al. (1967) and Greene et al. (1966). It differs from the Kunitz inhibitor by forming a much less stable complex: the inhibitor is eventually digested by the trypsin. The Kazal inhibitor is active on thrombin clotting activity but does not inhibit thrombin esterase activity nor does it act upon chymotrypsin, kallikrein, plasmin, urokinase (Burch et al. 1967) or human trypsin (Feeney et al. 1969). The intracellular basic trypsin inhibitor of Kunitz was first crystallized by Kunitz and Northrop in 1936. It has been shown by Anderer and Hornle (1966) to be identical with the kallikrein inhibitor of bovine lungs and parotid gland. Chauvet and Archer (1972) report on a preparation from bovine ovary. Basic pancreatic trypsin inhibitor (BPTI) forms a very stable 1:1 complex with bovine trypsin between pH 3 and 10 (Avineri-Goldman et al. 1967; Cole and Parthasarathy 1972), and also human trypsins (Figarela et al. 1974). The dissociation constant at pH 8.0 has been reported as 6 X 10-14 Vincent and Lazdunski 1972). Chymotrypsin is also inhibited by BPTI (Blow et al. 1972) but is bound less strongly. Chauvet and Archer (1975) indicate that Lys-15 is involved in the reaction with chymotrypsin. See also Engel et al. (1974). Imhoff and Keil-Dlouhá (1971) indicate that the "chymotryptic" active site participates in the reaction with trypsin. The inhibitor-chymotrypsin complex has been crystallized (Ruhlmann et al. 1971). Plasmin is inhibited by BPTI. Summaria et al. (1975) have reported on the plasmin-inhibitor complex. Reddy and Markus (1973) have shown that BPTI forms a ternary complex with plasminogen and streptokinase that is inactive. Spilberg and Osterland (1970) indicate that it inhibits the proteolytic activity of polymorphonuclear lysosomal lysates. Sardesai and Thal (1966) report that abnormal increase in plasma proteases of pancreatitis can be reduced to normal ranges but that the normal proteolytic activity of plasma is not inhibited. The esterolytic, proteolytic and elastolytic activities of porcine elastase are not inhibited by BPTI (Gertler and Feinstein 1971). BPTI is a single polypeptide chain of 58 amino acids, including six cysteines forming three disulfide bridges (Huber et al. 1971), and having a molecular weight of 6,500. It may exist under physiological conditions as a dimer (Trautschold et al. 1967). Structure, conformation and binding studies have been reported by Creighton (1975), Geratz et al. (1975), Gelin and Karplus (1975), Quast et al. (1975), Pershina and Hvidt (1974), Vincent et al. (9174), Wang and Kassell (1974), Karplus et al. (1973), Blow et al. (1972), Wilson and Laskowski (1971). Huber et al. (1971), Liu et al. (1971), Chauvet et al. (1966), Edelhoch and Steiner (1965), Kassell and Laskowski (1965), and Gallagher et al. (1992). The inhibitor has been reviewed by Trautschold et al. (1967). Kallikreins are proteases that specifically release from kininogen an α2-globulin fraction of the serum, and biologically active polypeptides such as bradykinin and kallikin (kinin 9 and kinin 10 respectively) (Kato and Suzuki 1970; Colman et al. 1969; Trautschold et al. 1966). The kinins are very active, even in nanogram quantities, causing smooth muscle stimulation, peripheral vasodilation, enhancement of capillary permeability and pain (Lim et al. 1969), symptoms manifest in hemorrhagic or surgical shock, pancreatitis, etc. The physiologic and pathologic roles of kinins have been reviewed by Kellermayer and Graham (1968). Much information on kallikrein and hypotensive peptides may be found in the Proceedings of the International Symposium on Hypotensive Peptides (October, 1965, Florence, Italy) edited by Erdos et al. (1966). Spillerg and Osterland (1970) observed an anti-inflammatory effect of BPTI in induced acute arthritis in rabbits.

BPTI is reported to be stable in acid or neutral milieu. Its isoelectric point is 10.5 (Trautschold et al. 1967). Activity of inhibitors is usually expressed in terms of substrate inhibited. In terms of a kallikrein inhibitor one K-I unit is that quantity which inactivates 2 kallikrein units being biologically determined. Trautschold indicates there to be 6000-7000 KI units per milligram of crystalline or chromatographically purified inhibitor.

Soybean Trypsin Inhibitor (Kunitz)

Soybean trypsin inhibitor (SBTI) first crystallized by Kunitz (1945) is one of several such inhibitors found in soybeans. (Fratalli 1969; Millar et al. 1969; Fratalli and Steiner 1968; Birk et al. 1967). The best known preparation is that of Kunitz. Steiner and Fratalli (1969) have reviewed the Kunitz and Bowman-Birk inhibitors. A protein (or polypeptide) proteinase inhibitor probably has peptide bonds compatible with the protease reactive site. Finkenstadt and Laskowski (1965 and 1967) and Ozawa and Laskowski (1966) indicate that a single Arg-Ile bond is cleaved by trypsin; a covalent bimolecular complex of inhibitor and trypsin results. On dissociation either virginal or modified inhibitor appears; see Hixson and Laskowski (1970a), Isheda et al. (1970) and Niekamp et al. (1969).

Molecular weight: 21,500 ± 800 (Wu and Scheraga 1972a).

Optimum pH: 7.0.

Composition: The Kunitz soybean inhibitor consists of a single polypeptide chain crosslinked by two disulfide bridges (Steiner 1965). Structural studies of the inhibitor and active site have been reported, Ellis et al. (1975), Woodward and Ellis (1975), Koide et al. (1974), Koide and Ikenaka (1973), Bidlingmeyer et al. (1972), Ikenaka et al. (1971), Papaioannou and Liener (1970), Hixson and Laskowski (1970), Kato and Tominager (1970), and Wu and Scherage (1962). Donovan and Beardslee (1975) have reported on the thermal denaturation of inhibitor complexes.

Isoelectric point: 4.5 (Kunitz 1947).

Specificity: Soybean inhibitor inhibits trypsin mole-for-mole and to a lesser extent chymotrypsin. (Bidlingmeyer et al. 1972). Lanchantin et al. (1969) report soybean inhibitor to form a one-to-one complex with beef or human thrombin thus blocking its specific proteolytic capacity to activate prothrombin. Nanninga and Guest (1964) report plasmin to be inhibited. STI has been reported to inhibit leukocytic proteases. (Lieberman and Gawad 1971), but not the esterolytic, proteolytic or elastolytic activities of porcine elastase (Gertler and Feinstein 1971).

Ovomucoid

Ovomucoids are the glycoprotein protease-inhibitors of avian egg white. There are several protease inhibitors in egg white. One acts upon ficin and papain (Fossum and Whitaker 1968); another, ovoinhibitor (Matsushima 1958), is a significant contaminant of crude ovomucoid preparations and acts upon bovine trypsin and chymotrypsin as well as porcine elastase and fungal proteinase (Feinstein and Gertler 1972; Gertler and Feinstein 1971; Liu et al. 1971; Tomimatsu et al. 1966). Chicken ovomucoid inhibits bovine trypsin mole for mole but does not inhibit human trypsin (Travis 1971; Feeney et al. 1969). It is electrophoretically heterogenous (Beeley and McCairns 1972; Beeley 1971; Melamed 1967; Bier et al. 1953). Both Bier and Beeley reported three major and two minor components with similar trypsin inhibiting activity but differing galactose, sialic acid and N-acetylglucosamine moieties. Ovomucoid conformation studies have been done by Murthy et al. (1973), Beeley (1972) and Donovan 1967). The arginyl residue for trypsin binding has been indicated by Liu et al. (1968). It does not contain tryptophan.

Molecular weight: The molecular weight is approximately 28,000 (Feeney et al. 1963). Davis et al. (1971) report it as 27,300 and Waheed and Salahuddin (1975) as 28,500 ± 3,500 for a distinct homogeneous variant.

Specificity: Feeney et al. (1967) have shown that although ovomucoid of different species show different inhibiting specificities, their physical properties differ only slightly. Thus, Rhodes et al. (1960) have shown that golden pheasant ovomucoid inhibits only chymotrypsin whereas that from turkey inhibits both trypsin and chymotrypsin simultaneously and duck ovomucoid inhibits two moles of trypsin and one of chymotrypsin simultaneously.

Lima Bean Trypsin Inhibitor

Lima bean trypsin inhibitor (LBI), which inhibits bovine as well as human trypsin (Feeney et al. 1969) and plasmin (Lewis and Ferguson 1953), acts upon both trypsin and chymotrypsin by forming equimolar complexes. The binding sites are distinct and independent (Krahn and Stevens 1972, 1971). The trypsin susceptible binding site is a lys-ser peptide bond (Krahn and Stevens 1972). The site of chymotrypsin action is a leu-ser bond (Krahn and Stevens 1970). As in other protease-inhibitors the "complexing" involves a reversible hydrolysis of the peptide bond (Krahn and Stevens 1973). See also Stevens and Doskoch (1973).

Lima bean trypsin inhibitors may be chromatographically separated into as many as six variants (Haynes and Feeney 1967). Jones et al. (1963) characterized four. All have similar but not identical amino acid composition, contain six or seven disulfide bonds and lack methionine and tryptophan. Molecular weights vary between 8,000-10,000. The complete amino acid sequence of component IV has been reported by Tan and Stevens (1971a and b). Krahn and Stevens (1972) report finding variations in activity of the four variants particularly with chymotrypsin, while essentially identical with respect to their trypsin inhibitory activity.

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