Trypsin inhibitor spectra

Figure 5.37 (a) Conventional phase-sensitive COSY spectrum of basic pancreatic trypsin inhibitor, (b) Double-quantum filtered (DQF) phase-sensitive COSY spectrum of the same trypsin inhibitor, in which singlet resonances and solvent signal are largely suppressed. Notice how clean the spectrum is, especially in the region near the diagonal line. (Reprinted from Biochem. Biophys. Res. Comm. 117, M. Ranee, et al., 479, copyright (1983) with permission from Academic Press, Inc.)... 

An additional emission band near 350 nm has been observed for lima bean trypsin inhibitor (LBTI).(173) The authors discussed both the possibility of contamination by tryptophan and excited-state tyrosinate formation. Since this 350-nm emission has a tyrosine-like excitation spectrum that is slightly shifted compared to that of the major 302-nm emission, it is also possible that the tyrosine residue in a fraction of the LBTI molecules could be hydrogen bonded. This model is supported by the observations that the phenol side chain is shielded from solvent and has an anomalously high pKa. 

Figure 4 shows a TOCSY spectrum with C(wi)-half-filter recorded with the small globular protein bovine pancreatic trypsin inhibitor (BPTI) using the pulse sequence of fig. 3. Although proton multiplets are usually difficult... 

The stress-70 proteins interact with a broad spectrum of polypeptide substrates, but they have some degree of specificity in their interactions. In several instances, it has been shown that a stress-70 protein can bind to proteins [e.g., bovine pancreatic trypsin inhibitor (BPTI), a-lactalbumin] that have been stabilized in a nonnative, or denatured, form by reduction and carboxymethylation of the cysteines that would normally form disulfides at the same time, they will not bind to the native forms of the same proteins (Liberek et al., 1991b Palleros et ai, 1991, 1992). This suggests that the peptide-binding activity of the stress-70 proteins discriminates in favor of polypeptides in a denatured, and possibly extended, conformation over those in a compact secondary and tertiary structure. NMR experiments demonstrating that the E. coli dnaK... 

For example, from protein basic pancreatic trypsin inhibitor (BPTI), four experimental ISNets are observed from a double quantum filtered COSY spectrum as shown at the bottom of Fig. 6. No one of them is completely matched onto its ISNet cluster center. 

Figure 2. 2D J-resolved proton spectrum of the protein bovine pancreatic trypsin inhibitor (BPTI). a. High-field region from 0.4—1.6 ppm, which contains the resonances of 19 methyl groups of the 360-MHz H NMR spectra of a 0.01 M solution of BPTI in D O at pH 4.5, 60°C. Prior to the Fourier transformation, the 2D data set was weighted in the t, and ts directions by weighting functions cos[(t.J 2Tx)ir]exp(tx/0.4Tx), with x = 1,2 Tj = 2.46 s, and Ts = 1.23 s are the maximum acquisition times in the ti and tj domains. The 2D J-resolved spectrum was computed from 64 X SI 92 data points and is presented as a (J, spectrum the top trace shows the conventional ID spectrum the bottom trace shows the projection of the 2D spectrum with 4> = rtl4. b. Presentation of the 2D J-resolved H spectrum (a) by cross sections. The resolved multiplets of 19 methyl protons are shown. The 2D resolved spectrum allows the analysis of otherwise overlapping multiplets, the accurate measurement of coupling constants, and the assignment of the resonances. (Reproduced, with permission, from Ref. 14. Copyright 1978, Academic...

Figure 6. Contour plot of a proton 2D NOE spectrum at 360 MHz of the basic pancreatic trypsin inhibitor. The protein concentration was 0.02 M solvent, HiO ...

Figure 5 Contour plot of the NOESY spectrum at 360 MHz of the basic pancreatic trypsin inhibitor. The protein concentration was 0.02 M, solvent D2O, pD = 3.8, T = 18°C. The spectral width was 4000 Hz 512 data points were used in each dimension 56 transients were accumulated for each value of U. The mixing time was 100 ms. The absolute value spectrum, obtained after digital filtering in both dimensions with a shifted sine bell, is shown. NOE connectivities for selected amino acid residues are indicated by the broken lines. Reproduced with permission of Academic Press from Kumar A, Ernst RR and Wuthrich K (1980) Biochemistry and Biophysics Research Communications 95 1.

Chromophoric inhibitor displacement.6 1 The spectrum of the dye proflavin changes significantly with solvent polarity. It is a competitive inhibitor of chymotrypsin, trypsin, and thrombin, and it undergoes a large increase in absorbance at 465 nm (Ae 2 X 104 M l cm 1) on binding (Figure 7.1). 

Schuitze first described AAT in 1955, later giving it this name because of its inhibition of trypsin. The name tti-proteinase inhibitor was later introduced because of AAT s broad spectrum of inhibition of serine proteases, not just trypsin. Both names are currently used, but AAT is preferred by most clinicians and clinical laboratorians and is used in this chapter. 

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