Studies on the reversible inactivation of papain and cathepsin

Abstract

THE natural activation of cathepsin and papain by sulphydryl compounds acquired increased physiological interest when it became known [Purr, 1933] that the combination, vitamin C-Fe", can activate not only arginase but also cathepsin. Later, Maschmann and Helmert [1934] found a similar activation of papain by the vitamin C-Fe" complex.The activation of these two proteinases by such widely dissimilar types of compounds made it of interest to investigate further the mechanism of these activations. The experiments here reported show much promise, contributing not only to our knowledge of the nature of intracellular proteolysis, but also to our understanding of the part played by vitamin C in the regulation of cellular metabolic processes.Since the initial activities of papain and cathepsin vary considerably for different preparations, it was necessary to devise methods for obtaining entirely inactive enzyme preparations. For comparative studies in enzyme activation, it was essential that these inactive preparations should retain the property of being completely reactivated upon addition of the proper activator.Reversible inactivators of papain. I. Alloxan inactivation and reactivation. Papain extract was prepared from a commercial Merck papain powder by 6 hours' extraction with 10 parts of water. After centrifuging, 4 ml. of the clear fluid were allowed to stand for 30 minutes at 300 and PH 7 with 40 mg. of cysteine hydrochloride dissolved in 4 ml. of water. To the thus fully activated enzyme solution were then added 80 mg. of alloxan in 4 ml. of water, and the reaction mixture was incubated for 1 hour at 30°and p11 7. The volume was brought to 20 ml., and the papain activity determined on 10 ml.; H2S was bubbled through the remaining 10 ml. for 1 hour at PH 7, and the degree of reactivation determined. (If cysteine be used for reactivation, it is necessary to use a large excess, 50 mg., because of the presence of excess alloxan.) The measurement of enzymic activity was carried out by adding 5 ml. of 8 % gelatin, 2 ml. of M acetate buffer of PH 5 and sufficient water to bring the volume up to 25 ml. 10 ml. of this were analysed at once for free amino-groups, and a second 10 ml. after 1 hour's incubation at 300. The increase of free amino-groups, determined by the Van Slyke method, constitutes a measure of the enzymic activity, the results being expressed in ml. of 0-05N KOH.

The Removal of Cartilage Matrix, in Vivo, by Papain

“…The mechanism by which this occurs is not known, but warrants further investigation. It has been reported that iodoacetamide does not cause destruction of the enzyme and that following precipitation of iodoacetamide-inactivated papain with alcohol, the activity may be restored almost to its original level with cysteine (8).…”

Macroporous Biodegradable Cryogels of Synthetic Poly(α-amino acids)

“…Cryogel discs (∼0.8 g in weight) were incubated with 0.25 mg·mL –1 papain solution in 0.05 mol·L –1 Tris-HCl buffer (pH 7.5) with 1 mmol·L –1 of EDTA-Na 2 and 5 mmol·L –1 of l -cystein as activators. The activity of the enzyme was determined by spectrophotometry, using BAPNA as a chromogenic substrate . The activity was held constant throughout the degradation period (22.6 ± 1.9 U·g –1 at 25 °C) by adding the required amount of fresh enzyme and a portion of freshly prepared buffer with activators each 24 h. Each time before the fresh enzyme was added, the sample was thrice washed by gentle shaking with an at least 20-times excess of deionized water.…”

“…Incubation was carried out with gentle shaking (100 rpm) at 37 °C. The degradation was quenched by intensively rinsing the samples with excess of water and adding 20 mL of 1 mmol·L –1 iodoacetic acid solution in water …”

“…The activity of the enzyme was determined by spectrophotometry, using BAPNA as a chromogenic substrate. 51 The activity was held constant throughout the degradation period (22.6 ± 1.9 U•g −1 at 25 °C) by adding the required amount of fresh enzyme and a portion of freshly prepared buffer with activators each 24 h. Each time before the fresh enzyme was added, the sample was thrice washed by gentle shaking with an at least 20-times excess of deionized water. Incubation was carried out with gentle shaking (100 rpm) at 37 °C.…”

“…The degradation was quenched by intensively rinsing the samples with excess of water and adding 20 mL of 1 mmol•L −1 iodoacetic acid solution in water. 51 The progress of the degradation was observed by determining the relative decrease in the storage modulus and the mass loss of the sample. The storage modulus of each disc sample was measured individually before starting the degradation and then at given time intervals for a period of 3 days.…”