Fluorescence depolarization and rotational modes of tyrosine in bovine pancreatic trypsin inhibitor

The fluorescence of bovine pancreatic trypsin inhibitor (BPTI) is due to one or more of its four tyrosine residues. Observations of the stationary polarization of the fluorescence over a large range of temperatures and viscosities permit the demonstration of at least three modes of tyrosine rotation, and perhaps an ultrafast fourth one. The slowest mode is one of motion of the whole molecule; the second, a much faster motion limited to an amplitude of 11 degrees, is not changed by quenching of the fluorescence through addition of citrate and is therefore ascribed to the motion of internal tyrosines of BPTI. The third mode of motion is faster still; it has an amplitude similar to that of the second and, being sensitive to citrate quenching, is attributed to the rotation of the external tyrosine residue. A residual depolarization corresponding to a rotational amplitude of 22 degrees is deduced by comparison of the polarizations of BPTI and tyrosine dissolved in 80% glycerol-water at -40 degrees C. It is in accord in amplitude with the picosecond tyrosine rotations predicted by Karplus and collaborators from molecular dynamics computer simulations, but it could also originate, in whole or in part, from electronic energy transfer among the tyrosines.     

NMR-detected order in core residues of denatured bovine pancreatic trypsin inhibitor

The NMR characteristics of [14-38]Abu, a synthetic variant of BPTI that is partially folded in aqueous buffer near neutral pH, support a model of early folding events which begin with stabilization of the nativelike, slow exchange core [Barbar, E., Hare, M., Daragan, V., Barany, G., and Woodward, C. (1998) Biochemistry 37, 7822-7833 (1)]. In partially folded [14-38]Abu, urea denaturation profiles for representative amide protons show that global unfolding is non-two-state and that core residues require a higher concentration of urea to unfold. Dynamic properties of pH-denatured [14-38]Abu and fully reduced and unfolded BPTI analogue were determined from heteronuclear NMR relaxation measurements at similar solution conditions. Differences at various sites in the polypeptide chain were evaluated from spectral density functions determined from T1, T2, and steady-state heteronuclear NOE data. Although denatured [14-38]Abu contains no persistent secondary structure, its most ordered residues are those that, in native BPTI, fold into the slow exchange core. The fully reduced analogue is significantly more mobile and shows less heterogeneous dynamics, but at 1 degree C, restricted motion is observed for residues in the central segments of the polypeptide chain. These observations indicate that there is a developing core or cores even in highly unfolded species. Apparently the effect of 14-38 disulfide on unfolded     

Interaction between the basic inhibitor of bovine pancreas and chymotrypsin and trypsin. Selective anthraniloylation of the maleylated inhibitor

The interaction between the maleylated basic pancreatic inhibitor, anthraniloylated on its lysine-15 residue, and chymotrypsin is studied by fluorescence intensity, fluorescence polarization, circular dichroism, circular polarization of fluorescence and sedimentation. These measurements show that the interaction takes place through the entrance of the anthraniloyl group into an asymmetric environment in which it is rigidly held. The dissociation constant of the complex is 2.5 × 10^?8m. The interaction between the modified inhibitor and trypsin takes place through a site which is not the anthraniloylated lysine-15 side-chain, yet not far from it.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) to human and bovine factor Xa. A thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human and bovine factor Xa (Stuart-Prower factor; EC 3.4.21.6) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to human and bovine factor Xa are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human and bovine factor Xa decrease, thus reflecting the acidic pK shift of the His57 catalytic residue from 7.1, in the free enzyme, to 5.2, in the proteinase-inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human and bovine factor Xa are: Ka = 2.1 x 10(5)M-1 (at 21.0 degrees C), delta G degree = -29.7 kJ/mol (at 21.0 degrees C), delta S degree = +161 entropy units (at 21.0 degrees C), and delta H degree = +17.6 kJ/mol (temperature-independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human and bovine factor Xa have been analysed in parallel with those of related serine (pro)enzyme/Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human and bovine factor Xa was related to the inferred stereochemistry of the proteinase/inhibitor contact region.     

Interaction of trypsin with a trypsin inhibitor from bovine colostrum]

Kinetics of trypsin association with trypsin inhibitor from colostrum (IC) was studied. The association rate constant is 3-10-5 M- minus 1 sec- minus 1 at pH 7,8, 25 degrees C. The rate constant for the complex dissociation was determined from the kinetics of the IC displacement from the complex with trypsin by a specific substrate and was found to be 5-10- minus 6 sec- minus 1 (pH 7,8; 25 degrees C). The equilibrium constant (Ki) was measured in a special experiment and was equal to 4-10- minus 12 M (p H 7,8; 25 degrees C). The similarity of this reaction and the association of trypsin with other protein inhibitors was discussed.     

Secretion and immunochemical properties of the trypsin inhibitor from bovine colostrum.

A trypsin inhibitor was isolated from bovine colostrum by affinity chromatography. Immunoelectrophoresis detected two immunogenic components in the isolated inhibitor, but only one of these was specific for the inhibitor; the other one was identical with an antigen present in liver, kidney, spleen, adrenal, thyroid, thymus, brain, ovarian, testicular and udder tissue and in bull seminal plasma. Using immunoabsorption and immunofluorescence it was shown that the antigens specific for the trypsin inhibitor of colostrum could be demonstrated only in the tissue of an udder that is secreting colostrum. The inhibitor is secreted by the secretory epithelium of the milk alveoli of the udder, during the period when the latter secretes colostrum. This inhibitor was not detected in the milk. Cross-reaction between antisera to colostral inhibitor and basic pancreatic inhibitor or seminal plasma inhibitors yielded negative results. Antiserum to bovine colostral inhibitor showed a positive reaction with inhibitor isolated from porcine colostrum.