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1. 1. The proteolysis of beef-heart lactic dehydrogenase by nagarse, trypsin, and chymotrypsin has been studied with respect to the action of he proteolytic enzymes on the yield of fluorescence and polarization of the fluorescence of the dehydrogenase. The effects of proteolysis on the enzymatic activity as well as the coenzyme binding properties of the lactic dehydrogenase have also been investigated. The three proteases have been found to produce fragmens which are different in their fluorescent characteristics. 3-Acetylpyridine-DPNH has been found to protect against the proteolysis of the lactic dehydrogenase by nagarse and trypsin, but not against the action of chymotrypsin. 2. 2. As a result of these studies it is concluded that under our conditions, no enzymically active dialyzable fragment, or fragment containing bound coenzyme was produced. Complete integrity of the enzyme appears necessary for both enzymic activity and coenzyme binding. 3. 3. The effects of several concentrations of urea on the enzymic activity and fluorescence properties of the beef-heart lactic dehydrogenase are also presented. It has been found that 8 M urea causes a complete loss of enzymic activity, a loss of coenzyme binding capacity, a marked decrease in the fluorescence of the enzyme, and a drop in the polarization of protein fluoresencen. After removal of urea by dialysis, both the enzymic activity and the coenzyme binding property are still lost. However, there is some change in the polarization of protein fluorescence after dialysis indicating the formation of a structure with a new configuration. 4. 4. The significance of the fluorescence changes in the lactic dehydrogenase induced by urea and the proteolytic enzymes are discussed with respect to the structure and function of the dehydrogenase. 5. 5. A change in the absorption and the fluorescence properties of free tryptophan has been observed when the amino acid is allowed to stand in water solution. EDTA will prevent this change. |
