Trypsin difference spectra

As already described the enzyme-modified 7S protein retains a high molecular weight like the native protein it is excluded by Bio-Gel P-150 which has an exclusion limit of 150,000 daltons. The specificity of rennin is such that it is easy to control and limit the extent of digestion. When the enzyme action is monitored by ultraviolet absorption it is apparent that the rennin action is quite different from that obtained with an enzyme such as trypsin (Figure 1). Thus the UV difference spectrum for the rennin-modified protein shows an initial unfolding of the 7S protein chains as indicated by a negative peak at 236 nm. As rennin action continued this negative peak was replaced by a positive peak at about 237 nm characteristic of an ordered secondary structure. 

Fig. 133. The rate of appearance of the difference spectrum and the rate of the B22-B23 Arg-Gly peptide bond hydrolysis in the digestion of 0.5% beef zinc-insulin at pH 8 by 0.01% trypsin at 23°C. (Laskowksi ef ol., 1960a).

The aim of this work was to evaluate the inhibition effect of ome Allium aqueous extracts on acrosin and trypsin activities, a new aspect in the wide spectrum of Allium compound activities. The antioxidant capacity, probably responsible for antithrombosis and antitumoral effects, was determined for different parts of Allium plants (bulb, green leaf, white stalk). The toxicity of crude extracts was also determined. 

Trypsin digests of both wild type HRV virus and the mutant were analyzed using MALDI-TOF and MALDI Fourier transform mass spectrometry (FTMS). For HRV, the mass spectra for both wild-type and mutant were identical except for one peptide occurring at mlz 4700. This corresponds to residues 187-227 in the wild type sequence. The corresponding peak in the mutant mass spectrum occurs at 4783.5 (Fig. 4, inset). This mass difference of 83 Da corresponds exactly to a mutation of a Cys to Trp residue and there are no other possible mutations that would be separated by 83 Da. Since there is only one Cys in the peptide 187-227 at position 199, the mutant can be localized as HRV14-Cysl99Trp, which contains a Trp at position 199 instead of Cys in the wild type. 

Figure 4 Mass spectrometry analysis of difference peptides from the dehydroascorbate modified p-globin subunit. Dehydroascorbate modified deoxy recombinant hemoglobin was prepared as described in Fig. 2. Trypsin digestion was performed as described in materials and methods and the p-globin N-terminal peptide (see upper panel Fig. 3) and the difference peptide (see lower panel Fig. 3) analyzed hy LC-MS. The spectrum in the upper panel represents the P-globin N-terminal peptide and the spectrum in the lower panel represents the difference peptide. A summary of the data is presented in Table 1.

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