In vivo experiments with Sprague-Dawley rats were conducted in order to explore the influence of Cu(2+), Zn(2+) as well as of the combinations of both on the activity of trypsin. The solutions of the trace elements were given per os, the animals were killed 30 min after the applications, and the activity of trypsin was determined in the juice of the small intestine by usingN (α)-benzoyl-L-arginine-p-nitroanilide (L-BAPA) as the substrate. The activity of trypsin depends on the concentration of the trace elements. When Cu(2+) ions are applied, there is a minimum activity at 10(-5) mol Cu(2+)/L and a maximum at 10(-4) mol Cu(2+)/L. When giving Zn(2+) ions, a minimum of trypsin activity is found at 10(-5) mol Zn(2+)/L and a maximum at 5×10(-6) mol Zn(2+)/L. On the whole, the trypsin activity is lower when the Cu(2+)/Zn(2+) combinations are applied compared to the addition of the single trace elements. On principle, a good conformity of the in vivo results was found with in vitro results.
In vitro experiments were carried out in order to investigate the influence of Cu++-ions on the tryptic hydrolysis of casein and soybean protein. The admixture of 10(-3) to 10(-2) Mol Cu++/l to the reaction preparations results in the activation of trypsin in both substrates. The further increase of the Cu++-concentration results in a decrease of the trypsin activity and, with casein as substrate, in trypsin inhibition. The effects are similar with the synthetic substrate N alpha-benzoyl-L-arginine-p-nitroamilide (L-BAPA), however, trypsin is already activated after the admixture of approximately 5 x 10(-6) Mol Cu++/l. Since the admixture of Cu++-ions in experiments with pepsin in similar concentrations as with trypsin also results in an activation, the question is being discussed whether the improved protein digestibility and the higher weight increase of pigs which received 250 ppm copper can be traced back to these effects.
Zusammenfassung
Die Hydrolyse des synthetischen Trypsinsubstrats Nα‐Benzoyl‐L‐arginin‐p‐nitroanilid (L‐BAPA) wurde unter Cu++‐Einfluß mit Hilfe von Zeit‐Umsatz‐Kurven beobachtet. Dabei ergab sich bei allen verwendeten Kupferkonzentrationen und Enzym‐Substrat‐Verhältnissen im Zeitintervall zwei bis zehn Minuten Inkubationsdauer ein linearer Kurvenverlauf. Deutlich wurden eine leichte Aktivierung von Trypsin durch geringe Kupferkonzentrationen sowie eine starke, nahezu totale Inhibierung bei Zulage hoher, aber nicht toxischer Mengen. Eine Beeinflussung der Proteinverdaulichkeit durch diese Effekte kann diskutiert werden.
The rate of hydrolysis of the synthetic trypsin substrate Nα-enzoyl-L-arginine-p-nitroanilide was determined from turnover rate curves, when the trace elements zinc, iron, cobalt, and nickel were added in the concentration range from 0.9 · 10–7 to 0.9 . 10–3 mol Men+/1. The enzyme substrate ratio was 1:50. An influence on the activity of trypsin, depending on the element used and on its concentration could be determined. Cobalt and iron accelerated the enzyme’s capacity of hydrolysis at all concentrations used, whereas addition of higher but not toxic amounts of zinc or nickel resulted in an inhibition.
Zusammenfassung
Es wurden in vitro‐Versuche mit Trypsin und mit Nα‐Benzoyl‐L‐arginin‐p‐nitroanilin als Substrat durchgeführt, um in Gegenwart der Spurenelemente Cu++, Zn++, Fe3+, Co++ und Ni++ die kinetischen Parameter Michaeliskonstante KM, Inhibitorkonstante Kj, Reaktionsgeschwindigkeit v und spezifische katalytische Aktivität b zu bestimmen. KM für Trypsin beträgt 8,5 · 10‐4 Mol/l. Die zugesetzten Spurenenlemente hemmen bzw. aktivieren im Konzentrationsbereich von 0,9 · 10‐7 bis 0,9 · 10‐3 Mol Men+/l Trypsin in Abhängigkeit von der Konzentration nicht kompetitiv. Den stärksten Einfluß auf die Trypsinaktivität haben Cu++ ‐Ionen. Ki bei Konzentrationen ≥ 0,9 · 10‐6 Mol Cu++/l wurde bestimmt zu 5,4 · 10‐6 Mol/l.
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