Planta
Planta (Berl.)131, 145-148 (1976)
9 by Springer-Verlag 1976
Cyanide Formation in Preparations from ChloreUa vulgaris Beijerinck: Effect of Sonication and Amygdalin Addition Hans-Siegfried Gewitz, Elfriede K. Pistorius, Helga Voss, and Birgit Vennesland Forschungsstelle Vennesland der Max-Planck-Gesellschaft, HarnackstraBe 23, D-1000 Berlin 33, Germany
Summary. A critical evaluation of a method for recovering H C N from cell extracts is presented. Since crude extracts often bind or metabolize H C N extensively, the H C N recovered by distillation at room temperature represents only the difference between production and consumption. Sonication leads to H C N release from the alga, Chlorella vulgaris Beijerinck. Illumination of extracts at high light intensity in oxygen, with added Mn 2 + and peroxidase, also stimulates H C N production. In both processes, the H C N is probably formed by oxidation of nitrogenous precursors. Chlorella extracts cause formation of H C N from added amygdalin. No evidence was found, however, for the presence of cyanogenic glycosides in the algae.
Introduction
Many plants have long been known to accumulate cyanogenic glycosides. These are 7-hydroxynitriles, stabilized by glycosylation of the hydroxyl group. Their chemistry and biogenesis have been reviewed (Conn, 1973). When the tissues of cyanogenic plants are crushed, cyanide is released by the action of the hydrolytic enzymes which make contact with the substrate. Cyanide has been shown to be a component of an inactive form of nitrate reductase found in Chlorella vulgaris (Lorimer et al., 1974). High O2 tension and high light intensity stimulate formation of the inactive enzyme, presumably by stimulating H C N production (Pistorius et al., 1976). The present study was directed toward the eventual identification of the precursor(s) of H C N in Chlorella.
Materials and Methods Biological Materials Horseradish peroxidase, purity grade I, was purchased from Boehringer, Mannheim; o-dinitrobenzene from Baker Chemicals N.V.,
Deventer, Netherlands; 4-nitrobenzaldehyde from Merck-Schuchardt, Hohenbrunn; and amygdalin from E. Merck, Darmstadt. ChlorelIa vuIgaris cells were grown in continuous white light on mineral salts medium with nitrate as the only source of nitrogen, in a stream of 5% (v/v) CO 2 in air at 2022~ as previously described (Vennesland and Jetschmann, 1971; Solomonson and Vennesland, 1972). Cells were harvested by centrifugation after 48 h of growth, and washed with water. French press extract of Chlorella cells was prepared as previously described from a cell suspension containing 250 gl cells per ml (Pistorius etal., 1976).
Experimental Procedures The sonication was carried out with a MSE 100-W Ultrasonic Disintegrator (Measuring and Scientific Equipment Ltd., London), with the large probe, at an amplitude of 5.5 6 microns. Sample volumes of 12-14ml were placed in a 25 ml beaker cooled in an ice bath. Sonication periods of 5 to 10 rain were interrupted by cooling periods so that the measured temperature of the extract did not rise over 20~ For the experiment of Table 1, Chlorella cells were suspended in water to give a cell concentration of 250 gl per ml. The HCN was collected by distillation in conical Warburg vessels of about 18 ml volume, with a side arm and a center trough (Warburg and Krippahl, 1958). The main compartment contained biological materials and the additions indicated in the legends of the tables and figures. The center trough contained 0.2 ml 0.1 N NaOH. The vessels were connected to manometers and shaken at 21~ in the dark or in red light (9,000 lux), as indicated, and for three hours unless otherwise specified. The HCN liberated is absorbed in the alkali. The three-hour period was chosen to maximize the yield (Pistorius et al., 1975). Incubation at 25~ rather than at 21~ resulted in lower total recovery of added HCN, although the rate of collection of HCN in the alkali in the center trough was decidedly faster at the higher temperature. The quantitative measurement of HCN was carried out by a slight modification of the procedure of Guilbault and Kramer (1966), as follows: 0.5 ml 0.088 N NaOH, 1.0 ml of 0.1 M o-dinitrobenzene in ethylene glycol monomethyl-ether and 1 ml of 0.2 M 4-nitrobenzaldehyde in ethylene glycol monomethyl-ether are mixed and equilibrated to 20~ After addition of 0.1 ml of the unknown HCN solution in 0.1 N NaOH, the incubation at 20~ is continued for 30 rain. Extinction is measured at 578 nm in a cuvette of 1 cm light path with an Eppendorf colorimeter, against a reagent blank. Figure 1 shows a typical calibration curve. The color formation for a given amount of HCN increases with the HCN concentration. Reproducibility of individual points in separate determinations is about + 10%. If the amount of HCN in 0.1 ml exceeds 1.5 nmol, the unknown samples are suitably diluted with 0.1 N NaOH.
146
H.-S. Gewitz et al. : Effect of Sonication on H C N Formation L
I
i
I
I
i
I
Results
Promotion of H C N Formation by Sonication E GO
r-
1.2
The experiment of Table 1 shows that sonication of Chlorella cells leads to H C N liberation, which increases approximately linearly with the time of sonication. Even after a total sonication time up to 100 rain, there was no indication that the reaction had been exhausted.
1.0
I---
.< m
0.8
(.9 Z
Planta (Berl.)131, 145-148 (1976)
9 by Springer-Verlag 1976
Cyanide Formation in Preparations from ChloreUa vulgaris Beijerinck: Effect of Sonication and Amygdalin Addition Hans-Siegfried Gewitz, Elfriede K. Pistorius, Helga Voss, and Birgit Vennesland Forschungsstelle Vennesland der Max-Planck-Gesellschaft, HarnackstraBe 23, D-1000 Berlin 33, Germany
Summary. A critical evaluation of a method for recovering H C N from cell extracts is presented. Since crude extracts often bind or metabolize H C N extensively, the H C N recovered by distillation at room temperature represents only the difference between production and consumption. Sonication leads to H C N release from the alga, Chlorella vulgaris Beijerinck. Illumination of extracts at high light intensity in oxygen, with added Mn 2 + and peroxidase, also stimulates H C N production. In both processes, the H C N is probably formed by oxidation of nitrogenous precursors. Chlorella extracts cause formation of H C N from added amygdalin. No evidence was found, however, for the presence of cyanogenic glycosides in the algae.
Introduction
Many plants have long been known to accumulate cyanogenic glycosides. These are 7-hydroxynitriles, stabilized by glycosylation of the hydroxyl group. Their chemistry and biogenesis have been reviewed (Conn, 1973). When the tissues of cyanogenic plants are crushed, cyanide is released by the action of the hydrolytic enzymes which make contact with the substrate. Cyanide has been shown to be a component of an inactive form of nitrate reductase found in Chlorella vulgaris (Lorimer et al., 1974). High O2 tension and high light intensity stimulate formation of the inactive enzyme, presumably by stimulating H C N production (Pistorius et al., 1976). The present study was directed toward the eventual identification of the precursor(s) of H C N in Chlorella.
Materials and Methods Biological Materials Horseradish peroxidase, purity grade I, was purchased from Boehringer, Mannheim; o-dinitrobenzene from Baker Chemicals N.V.,
Deventer, Netherlands; 4-nitrobenzaldehyde from Merck-Schuchardt, Hohenbrunn; and amygdalin from E. Merck, Darmstadt. ChlorelIa vuIgaris cells were grown in continuous white light on mineral salts medium with nitrate as the only source of nitrogen, in a stream of 5% (v/v) CO 2 in air at 2022~ as previously described (Vennesland and Jetschmann, 1971; Solomonson and Vennesland, 1972). Cells were harvested by centrifugation after 48 h of growth, and washed with water. French press extract of Chlorella cells was prepared as previously described from a cell suspension containing 250 gl cells per ml (Pistorius etal., 1976).
Experimental Procedures The sonication was carried out with a MSE 100-W Ultrasonic Disintegrator (Measuring and Scientific Equipment Ltd., London), with the large probe, at an amplitude of 5.5 6 microns. Sample volumes of 12-14ml were placed in a 25 ml beaker cooled in an ice bath. Sonication periods of 5 to 10 rain were interrupted by cooling periods so that the measured temperature of the extract did not rise over 20~ For the experiment of Table 1, Chlorella cells were suspended in water to give a cell concentration of 250 gl per ml. The HCN was collected by distillation in conical Warburg vessels of about 18 ml volume, with a side arm and a center trough (Warburg and Krippahl, 1958). The main compartment contained biological materials and the additions indicated in the legends of the tables and figures. The center trough contained 0.2 ml 0.1 N NaOH. The vessels were connected to manometers and shaken at 21~ in the dark or in red light (9,000 lux), as indicated, and for three hours unless otherwise specified. The HCN liberated is absorbed in the alkali. The three-hour period was chosen to maximize the yield (Pistorius et al., 1975). Incubation at 25~ rather than at 21~ resulted in lower total recovery of added HCN, although the rate of collection of HCN in the alkali in the center trough was decidedly faster at the higher temperature. The quantitative measurement of HCN was carried out by a slight modification of the procedure of Guilbault and Kramer (1966), as follows: 0.5 ml 0.088 N NaOH, 1.0 ml of 0.1 M o-dinitrobenzene in ethylene glycol monomethyl-ether and 1 ml of 0.2 M 4-nitrobenzaldehyde in ethylene glycol monomethyl-ether are mixed and equilibrated to 20~ After addition of 0.1 ml of the unknown HCN solution in 0.1 N NaOH, the incubation at 20~ is continued for 30 rain. Extinction is measured at 578 nm in a cuvette of 1 cm light path with an Eppendorf colorimeter, against a reagent blank. Figure 1 shows a typical calibration curve. The color formation for a given amount of HCN increases with the HCN concentration. Reproducibility of individual points in separate determinations is about + 10%. If the amount of HCN in 0.1 ml exceeds 1.5 nmol, the unknown samples are suitably diluted with 0.1 N NaOH.
146
H.-S. Gewitz et al. : Effect of Sonication on H C N Formation L
I
i
I
I
i
I
Results
Promotion of H C N Formation by Sonication E GO
r-
1.2
The experiment of Table 1 shows that sonication of Chlorella cells leads to H C N liberation, which increases approximately linearly with the time of sonication. Even after a total sonication time up to 100 rain, there was no indication that the reaction had been exhausted.
1.0
I---
.< m
0.8
(.9 Z
