Chymotrypsin toxicity

Selected entries from Methods in Enzymology [vol, page(s)] Types of organophosphorus inhibitors, 11,686-688 toxicity hazards, 11,688 purity and analysis, 11,688 solutions of organophosphorus compounds, 11,689 estimation of specific radioactivity of organophosphorus compounds, 11,689-690 method for estimating phosphorus content, 11,691 reactions with enzymes, 11,691 -701 [rate constants, 11,692 phosphorylation of chymotrypsin, 11, 694-696 identification of phosphoryl and phosphonyl peptides,... 

Because PMSE fails to inactivate acetylcholinesterase, this reagent is much less toxic than diisopropylfluoro-phosphate, and is also recommended as an alternative to the neurotoxic fluorophosphates and fluorophospho-nates. PMSE is freshly prepared as a 1-3 mM solution in water (higher concentrations will precipitate spontaneously). A better procedure is to first prepare a 20 mM PMSE solution in 2-propanol or dioxane this solution can then be added to the biological fluid with vortex mixing to achieve a 1-3 mM final concentration as a homogeneous solution. One should confirm that the alcohol or dioxane has little or no undesirable effect on enzymes or proteins of interest. See Chymotrypsin Protease Inhibitor Cocktails ... 

Contrary to the above-mentioned inhibitors, FK-448 (4-(4-isopropylpiperadinocarbonyl) phenyl 1,2,3,4,-tetrahydro-l-naphthoate methanesulfonate) is a low toxic as well as a potent and specific inhibitor of chymotrypsin. The effectiveness of this substance as an intestinal absorption enhancer has already been demonstrated in rats as well as in dogs. Coadministra-tion of FK-448 led to an enhanced absorption of insulin, which was monitored by a decrease in blood glucose level. The inhibition of chymotrypsin was found to be mainly responsible for the enhanced bioavailability [3]. Camostat mesilate (A,A -dimethyl carbamoylmcthyl-/)-(//-guanidino-benzoyloxy)phenylacetate methanesulfonate) [5] and Na-glycocholate [5,27] are further representatives of this class, exhibiting low toxicity. 

Biological properties such as the reactivity of enzymes, toxicity or drug action usually involve a combination of fundamental effects that give rise to equations which include more than one parameter. The Hansch equation is usually modified by the addition of terms such as steric parameters ( J or polar substituent constants (a). Examples are found in the catalytic action of a-chymotrypsin on 4-nitrophenyl esters of aliphatic acids (Equation 35). 

None of the protein inhibitors of NE presented in this chapter are protease specific. They all inhibit more than one protease, but they are protease class specific (Table 5). For example, ai-PI, SLPI, and eglin c are serine-protease inhibitors and inhibit trypsin, chymotrypsin, and cathepsin G in addition to NE. However, ai-PI is an inhibitor of neutrophil PR3, whereas SLPI and eglin c are only very weak inhibitors of PR3 [40]. By contrast, elafin, which shares 38% homology with the C-terminal domain of SLPI, does inhibit PR3. A strong selectivity for NE is important to reduce toxicity resulting from interference of the inhibitor with other proteolytic processes. 

Crown ethers also enhance the activity of enzymes in organic solvents.100 The enzyme is lyophilized in the presence of the crown ether before use. a-Chymotrypsin treated in this way with 18-crown-6 was 640 times as active in transesterification of amino acid esters as the untreated enzyme. However, the rate was still 50 times lower than in water. The use of surfactants to improve activity in organic solvents seems preferable because of a greater rate increase. They also avoid the toxicity and expense of the crown ethers. The cross-linked enzyme crystals also offer a way to obtain reasonable rates in organic solvents. 

Organophosphaies (OPs) and carbamates (CMs) are toxic compounds primarily due to inhibition of the acetylcholinesterase (AChE EC 3J.1.7) in the neurai and neuromuscular synapses. Buiyrylcholinesterase (BuChE EC 3.1.1.8) and other serine esterases, such as carboxylestcrase, trypsin, or chymotrypsin, arc also inhibited by these compounds. 

Important toxins are cobramine A and B from cobra toxin and crotactine and crotamine from crotox-in, the toxin of the North American rattlesnake. The toxic proteins are classified according to their mode of action cardiotoxins, neuFotoxins and protease inhibitors (with inhibitory activity toward chymotrypsin and trypsin). Cardiotoxins (heart muscle poisons) cause an irreversible depolarization of the cell membranes of heart muscle and nerve cells. Neurotoxins (nerve poisons) show curare-like activity they prevent neuromuscular transmission by blocking the receptors for the transmitters at the synapses of autonomic nerve endings and at the motor end plate of skeletal muscle. Protease inhibitors inhibit acetylcholine esterase and similar enzymes involved in nerve transmission. 

Besides chlorinated pesticides, some of the phosphorous pesticides are also chiral (see Table 2.1). The phosphorous pesticides also differ in their enantioselective toxicities. These pesticides were introduced in the 1950s to control insects in fruit, vegetables and other crops. Malathion is biotransformed into a racemic malaxon that has anti-acetylcholinesterate (insecticidal) activity. The /f-enantiomer has a 22 times greater inhibitory potency than the 5-enantiomer for bovine erythrocyte cholinesterase [39, 40]. The nerve agent, soman, has two chiral centres, and the two (—)-diastereoisomers are more potent inhibitors than their corresponding (-1-)-counteiparts for acetylcholinesterase and a-chymotrypsin. 

Ozoe Y, Mochida K, Eto M (1982) Reaction of toxic bicyclic phosphates with acetylcholinesterases and a-chymotrypsin. Agric Biol Chem 46 2527-2531... 

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