The influence of some methodological factors on measurement of tryptophan oxygenase activities in crude homogenates of rat liver

Abstract

1. With two different methods for assaying the tryptophan oxygenase activity in rat liver homogenates, the effects of some methodological factors on the activity of the enzyme were studied. 2. In fed, but not in starved, rats a compound(s) absorbing at 365 nm, interfering with the reading of kynurenine absorbance, disappeared gradually during incubation. 3. A correction for this tryptophan-independent reaction was necessary in order to determine correct tryptophan oxygenase activity. 4. Blood remaining in liver tissue post mortem can serve as a source of cofactor haem for tryptophan oxygenase, causing spuriously high values for the activity of the holoenzyme form of tryptophan oxygenase. 5. A rapid and progressive activation of tryptophan oxygenase post mortem occurs in undisrupted liver tissue, and this activation is temperature-dependent.

Determination of tryptophan oxygenase activity in rat liver homogenates

“…We find it hard to believe that this small difference could have such importance, since the main carbohydrate of our diet, starch, gives less marked 'blank' reactions immediately after food consumption than does intake of glucose and sucrose solutions (M0rland, 1974). In our study (Stowell & M0rland, 1983) we demonstrated that the blank reaction was most marked during the first period after the animals had finished eating during the dark period. Thus the phenomenon was reduced towards the end of the light period when the animals approached a more starved state.…”

“…This is not the standard procedure in the assay as performed by Badawy et al (1983), and is very time-consuming. We found that the variation could be reduced by replacing trichloroacetic acid with metaphosphoric acid (Stowell & M0rland, 1983) which has substantial buffer capacity at the desired endpoint pH. This allowed reproducible titration to be performed with a standard amount of NaOH for all samples run in the same incubation.…”

“…We have recently demonstrated that the assay of the liver enzyme tryptophan oxygenase (L-tryptophan: oxygen 2,3-oxidoreductase, EC 1.13.1 1.1 1) might be influenced by (1) a substrate-independent (blank) reaction interfering with the reading of kynurenine absorbance at 365 nm in samples from fed rats, (2) blood remaining in the liver affecting the amount of holoenzyme measured, and (3) rapid and progressive activation of the enzyme occurring post nmrtem in undisrupted liver tissue (Stowell & M0rland, 1983). Recently points (1) and (2) were subjected to some criticism (Badawy et al, 1983).…”

11–Deoxycortisol Induces Hepatic Tryptophan Oxygenase in Rats

“…heparin/ml saline) through the portal vein for 30 sec., and liver samples were frozen in liquid nitrogen 3'!2 min. after the perfusion and stored at -80" (Stowell & Msrland 1983).…”

“…Liver TO activity. TO activities were determined spectrophotometrically as described by Stowell & Msrland (1983), measuring the formation of kynurenine from tryptophan during the incubation of crude liver homogenates. The holoenzyme activity was determined by incubating the homogenates in the presence of 2.5 mM L-tryptophan, while the total enzyme activity was determined when the cofactor haem was present in excess in the incubation buffer (in the form of 2 FM haemoglobin) in addition to 2.5 mM L-tryptophan.…”