Effect of ultrasound on activation of serine proteases precursors.

The effect of ultrasound (US) (26.4 kHz, 26 W/cm2) on the activation process of a mixture of chymotrypsinogen and trypsinogen was studied. US led to a significant decrease in proteolytic activity, as well as inhibition of the activation process in general. It was shown that inhibition of proteinase activity under US influence was a consequence of inhibition of chymotrypsinogen-chymotrypsin transformation and the complete proteolytic trypsin degradation in the proenzymes mixture.

Application of pulse radiolysis to the study of proteins: chymotrypsin and trypsin.

The one-electron reduction of chymotrypsin, trypsin, and their zymogens have been studied by pulse radiolysis. The optical spectra of the transient products from the two active enzymes display a pH-dependent band at 360 nm, associated with the histidine-electron adduct. The yield of the histidyl radical as a function of pH is consistent with a pK(a) less than 4.5, which suggests that the radical is located at the enzyme active site. The histidines of the proenzymes chymotrypsinogen and trypsinogen are unreactive towards the hydrated electron. We conclude that formation of the histidine-electron adduct at the serine protease active site is sensitive to the physical alterations which accompany protease activation.

Gamma-irradiated chymotrypsin-like proteins. I. Structural changes.

The chymotrypsin-like proteins (chymotrypsin-CT,chymotrypsinogen-CTG, trypsin-T and modified chymotrypsins-at Met 192-MCT and at Tyr 146, 171-TCT), gamma-irradiated in the presence of air, were investigated. Irradiation leads to the unfolding of the native structure of CT-like proteins both in solution and in the dry state, which was shown by the tryptophan fluorescence, viscosimetry and microcalorimetry. The radiation yield of unfolded molecules Gconf was estimated and compared with (1) the rate constants for the reactions of OH-radicals with the proteins as determined by the p-nitrosodimethylaniline, (2) general stability of protein globule using the difference of the energies of the unfolded and globular conformations and (3) the radiation yield of tryptophan destruction in proteins-G-trp. There was a correlation between the values of Gconf and G-trp. The ratio G-trp/Gconf, which defines the number of destroyed tryptophan residues for one unfolded protein molecule, was constant within the limits of error. For CT, MCT, TCT and CTG, this ratio was on the average 3-2, and for T it was 2-2 residues. These facts point to the role of tryptophan destruction in the unfolding of the native structure of CT-like proteins on irradiation.

[Mechanism of the formation of paramagnetic centers in proteins exposed to UV-radiation]. / Mekhanizm obrazovaniia paramagnitnykh tsentrov v belkakh pri deistvii UF-sveta

It is shown that the process of formation of paramagnetic centres in UV-irradiated at 77degreesK protein solutions in a biquantum one. The protein molecule in triplet state is an intermediate product.

Photolysis accompanying peptide absorption in proteins. Electrophoresis and optical rotary dispersion studies.

Exposure of proteins and polypeptides to ultraviolet radiation below 240 nm produces peptide cleavage which may or may not be accompanied by observable changes in conformation and optical rotary dispersion (ORD) properties, depending on the stability of the secondary and tertiary structure of the macromolecule under the experimental conditions. Helical and coiled forms of poly-L-glutamic acid undergo degradation at similar rates but only the helical form shows a significant change in rotatory properties. The helical form of poly-L-lysine, but neither the coiled nor beta forms, shows a change in [alpha](233) on irradiation at 233 nm. beta-Lactoglobulin shows essentially no change in [alpha](233) on irradiation in either dilute salt solution or 4 M urea at room temperature; however, in 4 M urea at 56 degrees C a large change occurs. A model is developed which shows that studies of the effect of radiation on ORD properties may be useful in providing information on possible intermediate steps in protein denaturation. The method is illustrated with results on bovine plasma albumin. A quantum yield, 4.3 x 10(-3) moles/einstein, was obtained for peptide cleavage in this protein at 225 nm. These studies, based on gel electrophoresis, also showed that the fragments produced are essentially random, suggesting that transfer of energy from aromatic residues is not an important contributor to the peptide photolysis. Possible errors which could arise in ORD and other studies involving intense ultraviolet radiation are considered.