Optimization of post-column photolysis and electrochemical detection for the liquid chromatographic determination of 3-nitro-L-tyrosine.

A new post-column photolysis technology has been developed based on the use of a low pressure, low temperature UV lamp and TiO2 coated knitted reaction coil. As a test case the developed technique was used for the determination of 3-nitro-L-tyrosine by liquid chromatography with electrochemical detection. Different photolysis lamps and reactor tubing lengths were evaluated in terms of their effect on the separation efficiency and/or photolysis efficiency. A detection limit of 0.5 nM (10 fmol) for 3-nitro-L-tyrosine was achieved under optimized conditions, with a linear correlation coefficient of R2 = 0.9898 over a concentration range of 2-100 microM. Pre-injection photolysis of 3-nitro-L-tyrosine indicated that dihydroxyphenylalanine is the main photolysis product. In general, use of the photoreactor prior to liquid chromatography is an excellent method for exploring photodegradation products of an analyte in conjunction with the full range of available liquid chromatography detectors.

Refolding of serine proteinases.

Bovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%-50% at pH 8.6 and 4 degrees C, and the half-time for the refolding was approximately 60-75 min. We then refolded threonine-neochymotrypsinogen, which is a two-chain structure held together by disulfide bonds and produced on cleavage of Tyr 146-Thr 147 in native chymotrypsinogen [Duda CT, Light A, J Biol Chem 257 9866-9871, 1982]. Neochymotrypsinogen was denatured and fully reduced, and the thiols were converted to the mixed disulfide of glutathione. The two polypeptide fragments, representing the amino- and carboxyl-terminal domains, were separated on Sephadex G-75. Mixtures of the polypeptide fragments varying in the ratio of their concentration from 1:5 to 5:1 were refolded with yields of 21-28%. The lack of dependence on the concentration of either fragment and the relatively high yields suggest independent folding of the amino- and carboxyl-terminal domains. When the globular structures of the domains formed, they then interacted with one another and produced the native intermolecular disulfide bridge and the proper geometry of the active site.

Refolding of bovine threonine-neochymotrypsinogen.

The mixed disulfide derivative of fully reduced neochymotrypsinogen was refolded at pH 9.2 and 4 degrees C with 4 mM cysteine as the disulfide interchange catalyst. The yield of regenerated neochymotrypsinogen was 25%; the corresponding yield of refolded chymotrypsinogen was 50%. The refolded neochymotrypsinogen exhibited the characteristics of the native molecule as determined from polyacrylamide gel electrophoresis and the enzymatic properties of the activated zymogen. The rate of refolding of neochymotrypsinogen was approximately the same as that found for chymotrypsinogen. These studies show that two separate fully reduced polypeptide chains were capable of refolding, associating with one another, and regenerating a native structure with full biological activity.

Effects of gamma irradiation on chromatin activity of sugar beet tissue.

Chromatin-associated RNA polymerase activity increases during washing of sugar beet tissue to a maximum by 20 hours. This increase was inhibited by dosages of gamma irradiation between 50 and 400 krad. Template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, also increased with washing and was inhibited, although to a lesser extent, by the above irradiation dosages. Neither endogenous polymerase activity nor template availability was affected by high dosages (300 krad) in unwashed tissue. Exposure of tissue to irradiation (300 krad) at different times during a 20-hour washing period severely inhibited the development of RNA polymerase activity during the early stages of washing. The inhibition of template availability, however, was independent of time of irradiation. The data presented are discussed in relation to the mechanisms involved in the inhibitory effects of gamma irradiation on RNA production and subsequent protein synthesis.

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root.

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg(2+) or Mn(2+)) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.Washing tissue in solutions of gibberellic acid and auxin enhances template availability of the isolated chromatin. Experiments with isolated nuclei indicate an effect of these hormones on RNA synthesis.

Increased ribonucleic Acid polymerase activity associated with chromatin from internodes of dwarf pea plants treated with gibberellic Acid.

Gibberellic acid increases the level of RNA polymerase associated with chromatin isolated from expanding internodes of light-grown, dwarf pea plants (Pisum sativum L.), without a detectable increase in the amount of DNA template available.