Trypsin inhibitors

As examples of applications, we present the overall accuracy of predicted ionization constants for about 50 groups in 4 proteins, changes in the average charge of bovine pancreatic trypsin inhibitor at pH 7 along a molecular dynamics trajectory, and finally, we discuss some preliminary results obtained for protein kinases and protein phosphatases. 

The presented algorithm was applied to 4 proteins (lysozyme, ribonuclease A, ovomucid and bovine pancreatic trypsin inhibitor) containing 51 titratable residues with experimentally known pKaS [32, 33]. Fig. 2 shows the correlation between the experimental and calculated pKaS. The linear correlation coefficient is r = 0.952 the slope of the line is A = 1.028 and the intercept is B = -0.104. This shows that the overall agreement between the experimental and predicted pKaS is good. 

M. H. Hao, M. R. Pincus, S. Rackovsky, and H. A. Scheraga. Unfolding and refolding of the native structure of bovine pancreatic trypsin inhibitor studied by computer simulations. Biochemistry, 32 9614-9631, 1993. 

In periodic boimdary conditions, one possible way to avoid truncation of electrostatic interaction is to apply the so-called Particle Mesh Ewald (PME) method, which follows the Ewald summation method of calculating the electrostatic energy for a number of charges [27]. It was first devised by Ewald in 1921 to study the energetics of ionic crystals [28]. PME has been widely used for highly polar or charged systems. York and Darden applied the PME method already in 1994 to simulate a crystal of the bovine pancreatic trypsin inhibitor (BPTI) by molecular dynamics [29]. 

Brooks B and M Karplus 1983. Harmonic Dynamics of Proteins Normal Modes and Fluctuations in Bovine Pancreatic Trypsin Inhibitor. Proceedings of the National Academy of Sciences USA 80 6571-6575. 

Guo Z and C L Brooks III 1998. Rapid Screening of Binding Affinities Application of the A-Dynamics Method to a Trypsin-Inhibitor System. Journal of the American Chemical Society 120 1920-1921. 

Since feeds contain other substances than those required by the animals of interest, studies have also been conducted on antinutritional factors in feedstuffs and on the use of additives. Certain feed ingredients contain chemicals that retard growth or may actually be toxic. Examples are gossypol in cottonseed meal and trypsin inhibitor in soybean meal. Restriction on the amount of the feedstuffs used is one way to avoid problems. In some cases, as is tme of trypsin inhibitor, proper processing can destroy the antinutritional factor. In this case, heating of soybean meal is effective. 

Lupine seed, though used primarily in animal feeds (see Feeds AND FEED ADDITIVES), does have potential for use in human appHcations as a replacement for soy flour, and is reported to contain both trypsin inhibitors and hemagglutenins (17). The former are heat labile at 90°C for 8 minutes the latter seem much more stable to normal cooking temperatures. Various tropical root crops, including yam, cassava, and taro, are also known to contain both trypsin and chymotrypsin inhibitors, and certain varieties of sweet potatoes may also be impHcated (18). 

Arachin, the counterpart of glycinin in peanuts, consists of subunits of 60,000—70,000 mol wt which on reduction with 2-mercaptoethanol yield polypeptides of 41,000—48,000 and 21,000 mol wt (17) analogous to the behavior of glycinin. In addition to the storage proteins, oilseeds contain a variety of minor proteins, including trypsin inhibitors, hemagglutinins, and enzymes. Examples of the last are urease and Hpoxygenase in soybeans. 

Pea.nuts, The proteins of peanuts are low in lysine, threonine, cystine plus methionine, and tryptophan when compared to the amino acid requirements for children but meet the requirements for adults (see Table 3). Peanut flour can be used to increase the nutritive value of cereals such as cornmeal but further improvement is noted by the addition of lysine (71). The trypsin inhibitor content of raw peanuts is about one-fifth that of raw soybeans, but this concentration is sufficient to cause hypertrophy (enlargement) of the pancreas in rats. The inhibitors of peanuts are largely inactivated by moist heat treatment (48). As for cottonseed, peanuts are prone to contamination by aflatoxin. FDA regulations limit aflatoxin levels of peanuts and meals to 100 ppb for breeding beef catde, breeding swine, or poultry 200 ppb for finishing swine 300 ppb for finishing beef catde 20 ppb for immature animals and dairy animals and 20 ppb for humans. 

M Vasquez, ElA Scheraga. Calculation of protein conformation by the build-up procedure. Application to bovine pancreatic trypsin inhibitor using limited simulated nuclear magnetic resonance data. J Biomol Struct Dyn 5 705-755, 1988. 

The details of many all-atom unfolding simulation studies have been summarized in several reviews [17,46,47]. These studies include unfolding simulations of a-lactalbumin, lysozyme, bovine pancreatic trypsin inhibitor (BPTI), barnase, apomyoglobin, [3-lacta-mase, and more. The advantage of these simulations is that they provide much more detailed information than is available from experiment. However, it should be stressed that there is still only limited evidence that the pathways and intermediates observed in the nanosecond unfolding simulations correlate with the intermediates observed in the actual experiments. 

ST Russell, A Warshel. Calculations of electrostatic energies m proteins The energetics of ionized groups m bovine pancreatic trypsin inhibitor. J Mol Biol 185 389-404, 1985. 

Figure 2.14 shows examples of both cases, an isolated ribbon and a p sheet. The isolated ribbon is illustrated by the structure of bovine trypsin inhibitor (Figure 2.14a), a small, very stable polypeptide of 58 amino acids that inhibits the activity of the digestive protease trypsin. The structure has been determined to 1.0 A resolution in the laboratory of Robert Huber in Munich, Germany, and the folding pathway of this protein is discussed in Chapter 6. Hairpin motifs as parts of a p sheet are exemplified by the structure of a snake venom, erabutoxin (Figure 2.14b), which binds to and inhibits... 

Wlodawer, A., Deisenhofer, J., Huber, R. Comparison of two highly refined structures of bovine pancreatic trypsin inhibitor. /. Mol. Biol. 193 145-156, 1987. 

Weissman, J.S., Kim, P.S. Kinetic role of non-native species in the folding of bovine pancreatic trypsin inhibitor. Proc. Natl. Acad. Sci. USA 89 9900-9904, 1992. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

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