Surface-simulation synthesis of the substrate-binding site of an enzyme. Demonstration with trypsin.

From the X-ray co-ordinates of bovine trypsin and its complexes with substrate analogues (benzamidine) and with soya-bean trypsin inhibitor, a peptide (TP) was designed and synthesized by surface-simulation synthesis, a concept previously introduced by this laboratory, to mimic the binding site of trypsin. Also, a control peptide (CTP) was synthesized that contained all the amino acids present in the TP peptide, except that their order was randomized. The radioiodinated TP peptide bound specifically to adsorbents of benzamidine, whereas the control CTP peptide exhibited no binding activity. Conjugates to succinyl (3-carboxypropionyl)-lysozyme of the TP peptide, control CTP peptide and other unrelated peptides were examined by a radiometric binding assay for the ability to bind soya-bean trypsin inhibitor and human alpha 1-antitrypsin. Conjugates of the TP peptide exhibited considerable binding activity to adsorbents of soya-bean trypsin inhibitor or alpha 1-antitrypsin. None of the other peptide conjugates possessed any binding activity. Action of the active-site-directed reagents phenylmethanesulphonyl fluoride and di-isopropyl phosphorofluoridate on free TP and CTP peptides resulted in the modification of a serine residue in the TP peptide whereas the CTP peptide remained unaltered. The TP peptide, either in the free form or as a conjugate on succinyl-lysozyme, had no enzymic activity on protein substrates or on tosylarginine methyl ester. These findings indicated that the binding activity of an enzyme was well mimicked by the surface-stimulation peptide but that reproduction of the catalytic activity was not obtained.     

Two simple quantitative assays for trypsin inhibitors using immobilized trypsin.

Two new methods for quantitative assay of trypsin inhibitors, suitable for large numbers of samples, are described. The assay methods use trypsin-Sepharose conjugates incorporated into agarose gel slabs. Trypsin inhibitors are allowed to diffuse into, or are electrophoretically moved through, the slabs, and the consequent areas of inactivation of immobilized trypsin are visualized using a histochemical enzyme substrate. Quantitation of the trypsin inhibitor content of samples can be made on the basis of the inactivated areas. The limit of detection is 1–2 μg of soybean trypsin inhibitor and determinations are reproducible to 10% or better. Measured trypsin inhibitor contents of several legume species and varieties agree with spectrophotometric determinations.     

Characterization of agarose-bound trypsin.

Agarose-bound trypsin (EC 3.4.21.4) was prepared and its properties were compared with those of soluble trypsin. The bound form of the enzyme was found to be equally available to large and small molecular weight substrates as the soluble form. In addition, the bound form of the enzyme showed the same specificity towards protein substrates as the soluble enzyme. However, the agarose-bound trypsin showed greater stability than the soluble trypsin to denaturing conditions for prolonged period of time.     

Glutathione as an inhibitor of trypsin induced proteolysis

Glutathione has been shown to inhibit trypsin induced proteolytic activity. A concentration of 6 mM of glutathione was found to completely inhibit proteolysis of 3H-proline labelled underhydroxylated procollagen as a substrate, whereas a concentration of 2.1 mM of glutathione caused 50% inhibition of proteolysis. When azocoll was used as a substrate for trypsin 50% inhibition of proteolysis was achieved with 1.4 mM of glutathione, though a complete proteolytic inhibition was attained at 4 mM glutathione. The results suggest that glutathione may be playing an important role in protein metabolism in a variety of disease and stress states.     

Chromatin superstructure A study with an immobilized trypsin

Hen erythrocyte chromatin was treated with trypsin immobilized on collagen membranes and the unfolding of chromatin fiber was followed by light scattering at 90° and flow linear dichroism. Chromatin was found almost completely decondensed when the bulk of H1 and H5 was digested while H3 was still intact. Further digestion leading to degradation of both H3 and the rest of H1 and H5 accounted for no more than 10–15% of the total effect. When chromatin with trypsin-cleaved H1 and H5 was titrated with increasing amounts of spermidine it folded similarly to the control sample. This finding suggests that charge neutralization appears a likely mechanism for maintaining the structure of the 30 nm chromatin fiber by the C-terminal domain of H1 and H5.     

Adenylate cyclase stimulation by trypsin.

Adenylate cyclase activity of a rat embryo fibroblast cell line (F111) is markedly increased by brief treatment with 1:300 trypsin. The degree of stimulation depends upon the length of time the cells are treated and the concentration of trypsin. Crystalline trypsin produced a stimulation similar to that obtained with 1:300 trypsin. Further, the addition of soybean trypsin inhibitor blocked the stimulation of adenylate cyclase by 1:300 trypsin. Trypsin-treated adenylate cyclase responds to PGE1, but there is no increase over that of untreated enzyme. This result and the increase in fluoride-stimulated levels of activity suggest that the trypsin is acting upon the catalytic unit of the enzyme.     

Radioimmunoassay of human plasma trypsin.

A radioimmunoassay has been developed for the determination of human trypsin (3.4.21.4) in plasma. It allows the measurement of trypsin concentration in spite of the presence of plasma or pancreatic inhibitors. The human trypsin used as a standard and for labelling was isolated from pancreatic tissue and purified by affinity chromatography. The antiserum was obtained from guinea-pigs immunized with partially purified human trypsin. In the radioimmunoassay, the values of trypsin in serial dilutions of plasma were parallel to those of the standard curves. The assay was shown to be reproducible, sensitive and specific. However, the two antisera used did not distinguish between the enzyme and its proenzyme. In normal subjects, plasma values were found to be around 400 ng/ml. They were 10-40 times higher in patients with acute pancreatitis. The method appears to be much more specific for the diagnosis of acute pancreatitis than the current determinations of amylase and lipase activity.     

13C NMR studies of the binding of soybean trypsin inhibitor to trypsin.

NMR studies of the complex between trypsin and soybean trypsin inhibitor with 1-¹³C-arginine and modified inhibitor with 1-¹³C-lysine show that these complexes involve almost exclusively non-covalent binding of the inhibitor to the enzyme for trypsin/¹³C-Lys-inhibitor at pH 6.5 and 8.1 and for trypsin/¹³C-Arg-inhibitor at pH 5.0. At pH 7.1 for trypsin/¹³C-Arg-inhibitor both non-covalent and acyl enzyme forms are observed. Under no conditions did we observe evidence for a tetrahedral adduct between enzyme and inhibitor.     

Kinetics of interaction of trypsin with an anionic inhibitor of trypsin BWI-1a from buckwheat seeds

The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k(ass)) was 2.2 x 10(6) M-1 x sec-1 and the dissociation rate constant (k(off)) of the enzyme--inhibitor complex was 3.5 x 10(-3) sec-1; the inhibition constant Ki was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.