Gas-liquid chromatographic determination of sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate residues on soybeans and foliage, soil, milk, and liver.

Residues of sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate and its free acid are determined by treating a sample extract with diazomethane to convert the residues to methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate. This compound is purified by chromatography on Florisil and measured by electron capture gas-liquid chromatography. The method has been used on soybeans, soybean foliage, soil, milk, and liver. It can detect 0.01 ppm with recoveries of 70--75%.

Bimolecular (black) lipid membranes: study of lipid-protein interactions.

An experimental method has been devised for the study of the inter-action of bimolecular (black) lipid membrane and protein in which 8-anilino-1-naphthalenesulfonic acid is used as a fluorescent probe. The presence of phospho-lipid in the membrane is necessary for the enhanced fluorescence.

A comparison of the effects of ultraviolet and ionizing radiations on trypsin activity and on its constituent amino acids.

Photons of 254nm. u.v. light, (60)Co gamma-rays and 1Mev electrons produce different patterns of destruction of individual amino acids in dried films of trypsin and in the corresponding amino acid mixture. For example, in the amino acid mixture u.v. light destroys tyrosine, tryptophan and cystine, whereas in trypsin only cystine is disrupted but with 10 times the initial yield. Further, in the amino acid mixture loss of half-cystine is a simple exponential function of dose, but in trypsin there appear to be two exponential components of the loss with yields that differ by a factor of 35. Both the gamma-rays and electrons destroy half-cystine, tryptophan, histidine and methionine in the amino acid mixture with remarkably high yields, whereas in trypsin doses that destroy almost all of the enzymic activity produce no detectable destruction of amino acid residues. These marked differences between the two preparations show that the radiation-sensitivity of a given amino acid alone and in a protein is different, and suggests that in trypsin there is fairly extensive migration of energy, charge or both with localization of damage at specific sites determined by this enzyme's internal organization. All three types of radiation produce appreciable amounts of ;damaged' (not completely inactivated) molecules which are prevented from reassuming an active configuration by the addition of 5.5m-urea; thiol reagents have a similar effect after bombardment with u.v. light or electrons. The patterns of destruction produced by gamma-rays and by electrons in both the amino acid mixture and in trypsin are different (some of the yields vary by a factor of 30). This result appears to be inconsistent with the popular belief that most of the energy absorbed from gamma-rays is associated with very-high-energy electrons.

Photochemical yields in ribonuclease and oxidized glutathione irradiated at different wavelengths in the ultraviolet.

The quantum yields for the disruption of various amino acids in glutathione and ribonuclease by 229, 254, 265, and 280 nm UV photons have been determined. The results of the measurements on the destruction of tyrosine and histidine and the loss of enzymic function in RNAse and the disruption of cystine in both compounds lead to the following conclusions: (a) The photodestruction of some and perhaps many constituent amino acid residues does not cause RNAse inactivation. (b) Contrary to the basic premise of proposals made by other authors, the photochemical yields of constituent residues in a protein are not the same as that for the same amino acids in solution alone-the difference is a function of the exciting wavelength. Further, the extent of histidine destruction varies by a large factor among three proteins. (c) Consistent with previous predictions, the present results show that photons absorbed in the aromatic residues of RNAse cause the disruption of cystines elsewhere in the enzyme. (d) Although cystine disruption appears to be the most prevalent mode of RNAse inactivation by photons of the four wavelengths studied, some of the minor mechanisms leading to loss of enzymic function may vary with the UV energy.

Vacuum ultraviolet studies on the nature of the radiation inactivation of trypsin.

Trypsin, in powder form and in frozen D(2)O-glucose solutions, at temperatures from 100 degrees to 300 degrees K, was excited with vacuum ultraviolet and near ultraviolet radiation to determine how absorbed photon energy is partitioned into radiative, nonradiative and/or inactivating processes; at 300 degrees K most of the absorbed energy is not reemitted, so that it (0.98-0.99 for excitation at 120 nm and 0.75-0.90 at 280 nm) is potentially available for inactivation. Since the effects of excitation wavelength and temperature on the emission quenching yields are generally different from those on the inactivation yields of dry trypsin, the mere retention of quenched energy by an enzyme does not necessarily lead to its inactivation. Thus, as predicted previously, the radiation inactivation of trypsin must proceed by rather specific mechanisms which undoubtedly depend upon environment-sensitive processes, since the nature of the molecular environment can modify the partitioning of energy so significantly; for example, there are differences in the phosphorescence-to-fluorescence ratio, in the activation energy for quenching, and in the lifetimes and kinetics of the decay of phosphorescence when trypsin in frozen glasses and dry trypsin are excited by various wavelengths of ultraviolet radiation.