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Some fluorescent substances contained in the marine “blood worm” (Thoracophelia mucronata)
Some Fluorescent Substances Contained in the Marine “Blood Worm” (Thoracophelia mucmnata) Denis L. Fox, B. Kenneth Koe,, F. J. Petracek and L. Zechmeister From the Gates and Crellin Laboratories of Chemistry, California Institute Technology, Pasadena, Calijornialrl; and the Scripps Institution oj Oceanography, University of California, La Jolla, California8
of
Received March 31, 1952
Nutrition and biochromes of the bright red, hemoglobin-containing marine polychaete worm, Thoracophelia mucronata, have been studied recently by Fox, Crane, and McConnaughey (1). The animals, measuring 30-50 mm. in length when adult, live buried in the intertidal sand which they ingest in great quantities. Upon extraction of the worms with lipoid solvents, followed by chromatographic analysis, carotenoid pigments and several intensely bluish fluorescent zones were obtained. The latter exhibited chiefly single extinction maxima in light petroleum at 310, 306, 305, 302.5, and 300 mp, respectively.4 Since, in the above study, spectral characteristics of the fluorescent substances from one particular lot of the worms (termed “sample 5”) were recorded (here referred to as sample ‘?I?‘), we have explored to what, degree the fluorescent materials of the animals may be subject to variations. For this purpose two further, independently collected samples (“II” and “III”), each weighing 10-11 kg., were studied. EXPERIMENTAL The collection of marine blood worms is best accomplished in the following way : Since they are small, buried in the damp beach sand for depths of from 2 to 12 in.
1 Contribution No. 1712. 2 This work was carried out on Contract NR-059-207 of the U. S. Office of Naval Research. a Contributions from the Scripps Institution of Oceanography, University of California, New Series No. 582. 4 The shorter wavelength regions were not explored because of lack of ultraviolet equipment at the time. 135
13G
FOX,
KOE,
PETRACEK
AND
ZECHMEISTER
(depending upon physical conditions), and are required in great numbers, the sand in which they are living is shoveled into a screening device. For this purpose, we have found it useful to employ a plastic screen, 18 meshes/in., backed by a coarse galvanized wire screen or so-called “hardware cloth”, to provide adequate support. These screens are in turn fastened to a cedar frame 36 in. long, 16 in. wide and 4 in. deep, which is supported above the sand at a height of from 1 to 3 ft. The mass of material on the screen is washed copiously with sea water to remove the sand which passes readily through the meshes. Contaminating organisms such as the sand crab Emerita analoga, the sand flea Orchestia traskiana, and bits of marine plant detritus, are removed by hand either directly from the screen or after placing the collected worms into a bucket and rapidly swirling the contents in fresh sea
250
300 mFL FIG. 1. Extinction curves in hexane: -, crystalline steroid isolated from Thoracophelia mucronata; - - -, ergosterol. water, which brings the contaminants to the top of the mass. For further refinement in segregating the animals, in the event that but limited numbers are required, we leave the mass of worms on a dampened cloth or paper, shielding half of the assembly from light. In from several hours to an overnight period, most of the active, healthy members of the collection will have crawled out onto the darkened damp surface, thus cleaning their exteriors of adhering sand, and incidentally voiding much additional sand from the gut. The average dry weight of the total organic matter in the worms was determined to be 17.70/,, the true ash 2.0%, and the sand contained in the gut 5.6%. Considering the manner of preparation of the material for shipment (rinsing most
FLUORESCENT
SUBSTANCES
IN
MARINE
BLOOD
WORM
137
of the sand away with water but leaving some adhering to the mucus) and assuming that the gut is filled with sand, the total amount of the sand in”the samples used was estimated to be about lO-12%. Eleven kg. of the worms (sample II) was kept under acetone during transportation. After decantation, the material was ground in Waring blendors with fresh acetone to a fine slurry which was spun in a basket centrifuge lined with filter paper. The residue was extracted three times with hexane-acetone mixtures
E
0.5
250
300
350 mp
FIG. 2. Extinction curve in hexane of fluorescent fraction “M” ex Thoracophetia (sample II); the maxima located at the following wavelengths coincide with those typical for fluoranthene: 236, 275,?87,302, 321, 336, and 358 ~JL (1: 1,2: 1, and 3: 1). To the combined extracts benzene and excess water were added cautiously, and the lower phase was discarded. The upper one was combined with a benzene-hexane (1: 1) extract of the acetone used originally to cover the worms plus that used in the blendors as mentioned. The total extract was concentrated in vacua to 1 1. and the dark-brown, strongly bluish-grey fluorescing liquid was saponified by exposure to 0.5 1. of 20% KOH in methanol for 3 day. After the addition of water the benzene-hexane solution layer was washed alkali-free, dried
138
FOX,
KOE,
PETRACEJK
AND
ZECHMEISTER
with sodium suifate, and filtered with suction through a layer of Celite in order to eliminate particulate contaminants. This purification was compIeted by addition of excess acetone; the resulting precipitate formed was removed, washed with acetone, and discarded. The solution was then washed acetone-free and again dried. Concentration iv vacua yielded 100 ml. of a dark-red, intensely blue-fluorescent concentrat,e which was developed on a 30 times 8 cm. alumina-caIcium
:I
E
250
400 mp
FIG. 3, Extinction curves in hexane of fluorescent fractions ez Thoracophelia (sample II): -, fraction A;- - -, B;-.-.-a, C;-'-I--', D. In Figs. 2-5 the alphabetic order A-O designates decreasing adsorption affinities on alumina-calcium hydroxide-Celite 3:l:f (developer, hexane containing O-50% benzene). hydroxide-Celite column (3:l: 1) with a 1:3 benzenehexane layer was stamped on atop the column to ietard the downward gummy contaminants and thus prevent a possibIe split of the In the following chromatogram the left figures designate , respective zones, in millimeters: 10 colorless (sugar layer).
mixture. A sugar migration of any main column. the width of the
FLUORESCENT
SUBSTANCES
IN
MARINE
BLOOD
48 yellow, orange and red zones; light brown to throughout this section. 7 yellowish-green fluorescence with a yellow-orange 55 orange; bluish fluorescence. 180 colorless, very weak fluorescence at bottom. Second filtrate fraction: colorless; blue fluorescence. First filtrate fraction: colorless; blue fluorescence.
139
WORM
lolet
fluorescence
pigment
streak.
i
250
FIG. 4. Extinction (sample
300
350 * mp
curves in hexane of fluorescent II): - - -, E; -‘-I-‘, F; -.-.-.,
fractions G; -,
ex Thoracophelia H.
Each of the adsorbed sections was cut out, eluted with acetone, transferred into hexane with water, washed, dried with sodium sulfate, and submitted to a systematic chromatographic fractionation in ultraviolet light. The details of these lengthy operations have been described by Petracek (3) as well as Koe(2) and will not be given here. Finally, about fifteen reasonably well separated zones were obtained (cf. legends to Figs. 2-5). Although a duplication of certain zones could not be absolutely excluded in a few instances, the individual nature of most of the components has been secured. Their adsorption affinities varied within
140
FOX, KOE, PETRACEK AND ZECHMEISTER
broad limits: Some were rapidly washed through an alumina-lime-Celite column with such a weak developer as pure hexane, while others required the addition of 5-50% benzene to the hexane in order to be clearly developed and differentiated. In still other instances a strong developer such as pure benzene had to be applied. The 4%mm. zone (see above) also yielded about 3 g. of a crystalline sterol aa a by-product, the extinction curve of which appears in Fig. 1, compared with that of ergosteroLs
E
250
330
350 mp
5. Extinction curves in hexane of fluorescent fractions (sample II) : -‘-.-I, 1,. -, J; -.-._. , K; - _ -, L; -‘-‘e’,
FIG.
The worm sample III (10 kg.) was worked methanol in place of acetone [cf. (3)].
up in a similar
ez Thoracophelia N; . . . . . . , 0. manner,
but using
RESULTS
With reference to their chromatographically homogeneous, fluorescent constituents, the three blood worm samples investigated turned out to 6 Although the two curves in Fig. 1 are almost identical, and the composition of this substance was found to be C&H,rO, the Carr-Price reaction product gave a spectral curve which was very different from those obtained from either e%gosterol or a crude commercial sample of 7-dehydrocholesterol.
FLUORESCENT
SUBSTANCES
IN
MARINE
BLOOD
WORM
141
be quite different as shown in Figs. 2-5 (er sample II) and in Fig. 6 (ez sample III). Moreover, none of these batches yielded the fluorescent compounds described earlier by Fox, Crane, and McConnaughey (1). Furthermore, samples II and III differed very widely from each other. The maximum extinction values for the numerous individual compounds were mostly observed in the region 220 to 280 rnp (in hexane). Figure 2 illustrates a special feature, viz. the occasional presence of aromatic polycyclic substances in the worms. The wavelength position of several (but not all) maxima of this fraction coincides with those typical for fluoranthene, a hydrocarbon which among others was recently identified in some barnacle extracts (4). E
400
300
v
FIG. 6. Extinction curves in hexane of fluorescent fractions and of carotenoid pigments (I-4) ez Z’horacophelia (sample III). (These fractions were separated chromatographically on calcium hydroxide-Celite 2:l by developing with hexane containing 04% acetone.)
Evidently, in accordance with the great variety of organic materials adsorbed to the ingest.ed sand, the marine blood worm is able to take up, and perhaps to store to a certain extent, fluorescent compounds of a wide chemical variety. In this connection there may be some significance to the demonstrated fact (1) that the worm not only fails to assimilate, but seems to degrade or otherwise alter the copious xanthophylls which greatly exceed the quantities of carotenes in its natural diet. SUMMARY
Extracts of two large samples (10-11 kg.) of the marine annelid worm Thoracophelia mwonata were examined, by means of chromatographic
142
FOX,
KOE,
PETK4CEK
AND ZECHMEISTER
fractionation in ultraviolet light, for fluorescing substances. A great variety and variability of such compounds were found to be present in the worms, collected at different times from the same habitat.
1. Fox, D. L., CRANE, S. C., AND MC~ONNAUGHEY, B. H., J. IMarine Research 7, 567 (1948). 2. KOE, B. K., Ph. D. Thesis, California Institute of Technology, Pasadena, Calif., 1952. 3. PETRACEK, F. J., M.S. Thesis, California Institute of Technology, Pasadena, Calif., 1951. 4. ZECHMEISTER, L., AND KOE, B. K., Arch. Biochem. Biophys. 36, 1 (1952).
of
Received March 31, 1952
Nutrition and biochromes of the bright red, hemoglobin-containing marine polychaete worm, Thoracophelia mucronata, have been studied recently by Fox, Crane, and McConnaughey (1). The animals, measuring 30-50 mm. in length when adult, live buried in the intertidal sand which they ingest in great quantities. Upon extraction of the worms with lipoid solvents, followed by chromatographic analysis, carotenoid pigments and several intensely bluish fluorescent zones were obtained. The latter exhibited chiefly single extinction maxima in light petroleum at 310, 306, 305, 302.5, and 300 mp, respectively.4 Since, in the above study, spectral characteristics of the fluorescent substances from one particular lot of the worms (termed “sample 5”) were recorded (here referred to as sample ‘?I?‘), we have explored to what, degree the fluorescent materials of the animals may be subject to variations. For this purpose two further, independently collected samples (“II” and “III”), each weighing 10-11 kg., were studied. EXPERIMENTAL The collection of marine blood worms is best accomplished in the following way : Since they are small, buried in the damp beach sand for depths of from 2 to 12 in.
1 Contribution No. 1712. 2 This work was carried out on Contract NR-059-207 of the U. S. Office of Naval Research. a Contributions from the Scripps Institution of Oceanography, University of California, New Series No. 582. 4 The shorter wavelength regions were not explored because of lack of ultraviolet equipment at the time. 135
13G
FOX,
KOE,
PETRACEK
AND
ZECHMEISTER
(depending upon physical conditions), and are required in great numbers, the sand in which they are living is shoveled into a screening device. For this purpose, we have found it useful to employ a plastic screen, 18 meshes/in., backed by a coarse galvanized wire screen or so-called “hardware cloth”, to provide adequate support. These screens are in turn fastened to a cedar frame 36 in. long, 16 in. wide and 4 in. deep, which is supported above the sand at a height of from 1 to 3 ft. The mass of material on the screen is washed copiously with sea water to remove the sand which passes readily through the meshes. Contaminating organisms such as the sand crab Emerita analoga, the sand flea Orchestia traskiana, and bits of marine plant detritus, are removed by hand either directly from the screen or after placing the collected worms into a bucket and rapidly swirling the contents in fresh sea
250
300 mFL FIG. 1. Extinction curves in hexane: -, crystalline steroid isolated from Thoracophelia mucronata; - - -, ergosterol. water, which brings the contaminants to the top of the mass. For further refinement in segregating the animals, in the event that but limited numbers are required, we leave the mass of worms on a dampened cloth or paper, shielding half of the assembly from light. In from several hours to an overnight period, most of the active, healthy members of the collection will have crawled out onto the darkened damp surface, thus cleaning their exteriors of adhering sand, and incidentally voiding much additional sand from the gut. The average dry weight of the total organic matter in the worms was determined to be 17.70/,, the true ash 2.0%, and the sand contained in the gut 5.6%. Considering the manner of preparation of the material for shipment (rinsing most
FLUORESCENT
SUBSTANCES
IN
MARINE
BLOOD
WORM
137
of the sand away with water but leaving some adhering to the mucus) and assuming that the gut is filled with sand, the total amount of the sand in”the samples used was estimated to be about lO-12%. Eleven kg. of the worms (sample II) was kept under acetone during transportation. After decantation, the material was ground in Waring blendors with fresh acetone to a fine slurry which was spun in a basket centrifuge lined with filter paper. The residue was extracted three times with hexane-acetone mixtures
E
0.5
250
300
350 mp
FIG. 2. Extinction curve in hexane of fluorescent fraction “M” ex Thoracophetia (sample II); the maxima located at the following wavelengths coincide with those typical for fluoranthene: 236, 275,?87,302, 321, 336, and 358 ~JL (1: 1,2: 1, and 3: 1). To the combined extracts benzene and excess water were added cautiously, and the lower phase was discarded. The upper one was combined with a benzene-hexane (1: 1) extract of the acetone used originally to cover the worms plus that used in the blendors as mentioned. The total extract was concentrated in vacua to 1 1. and the dark-brown, strongly bluish-grey fluorescing liquid was saponified by exposure to 0.5 1. of 20% KOH in methanol for 3 day. After the addition of water the benzene-hexane solution layer was washed alkali-free, dried
138
FOX,
KOE,
PETRACEJK
AND
ZECHMEISTER
with sodium suifate, and filtered with suction through a layer of Celite in order to eliminate particulate contaminants. This purification was compIeted by addition of excess acetone; the resulting precipitate formed was removed, washed with acetone, and discarded. The solution was then washed acetone-free and again dried. Concentration iv vacua yielded 100 ml. of a dark-red, intensely blue-fluorescent concentrat,e which was developed on a 30 times 8 cm. alumina-caIcium
:I
E
250
400 mp
FIG. 3, Extinction curves in hexane of fluorescent fractions ez Thoracophelia (sample II): -, fraction A;- - -, B;-.-.-a, C;-'-I--', D. In Figs. 2-5 the alphabetic order A-O designates decreasing adsorption affinities on alumina-calcium hydroxide-Celite 3:l:f (developer, hexane containing O-50% benzene). hydroxide-Celite column (3:l: 1) with a 1:3 benzenehexane layer was stamped on atop the column to ietard the downward gummy contaminants and thus prevent a possibIe split of the In the following chromatogram the left figures designate , respective zones, in millimeters: 10 colorless (sugar layer).
mixture. A sugar migration of any main column. the width of the
FLUORESCENT
SUBSTANCES
IN
MARINE
BLOOD
48 yellow, orange and red zones; light brown to throughout this section. 7 yellowish-green fluorescence with a yellow-orange 55 orange; bluish fluorescence. 180 colorless, very weak fluorescence at bottom. Second filtrate fraction: colorless; blue fluorescence. First filtrate fraction: colorless; blue fluorescence.
139
WORM
lolet
fluorescence
pigment
streak.
i
250
FIG. 4. Extinction (sample
300
350 * mp
curves in hexane of fluorescent II): - - -, E; -‘-I-‘, F; -.-.-.,
fractions G; -,
ex Thoracophelia H.
Each of the adsorbed sections was cut out, eluted with acetone, transferred into hexane with water, washed, dried with sodium sulfate, and submitted to a systematic chromatographic fractionation in ultraviolet light. The details of these lengthy operations have been described by Petracek (3) as well as Koe(2) and will not be given here. Finally, about fifteen reasonably well separated zones were obtained (cf. legends to Figs. 2-5). Although a duplication of certain zones could not be absolutely excluded in a few instances, the individual nature of most of the components has been secured. Their adsorption affinities varied within
140
FOX, KOE, PETRACEK AND ZECHMEISTER
broad limits: Some were rapidly washed through an alumina-lime-Celite column with such a weak developer as pure hexane, while others required the addition of 5-50% benzene to the hexane in order to be clearly developed and differentiated. In still other instances a strong developer such as pure benzene had to be applied. The 4%mm. zone (see above) also yielded about 3 g. of a crystalline sterol aa a by-product, the extinction curve of which appears in Fig. 1, compared with that of ergosteroLs
E
250
330
350 mp
5. Extinction curves in hexane of fluorescent fractions (sample II) : -‘-.-I, 1,. -, J; -.-._. , K; - _ -, L; -‘-‘e’,
FIG.
The worm sample III (10 kg.) was worked methanol in place of acetone [cf. (3)].
up in a similar
ez Thoracophelia N; . . . . . . , 0. manner,
but using
RESULTS
With reference to their chromatographically homogeneous, fluorescent constituents, the three blood worm samples investigated turned out to 6 Although the two curves in Fig. 1 are almost identical, and the composition of this substance was found to be C&H,rO, the Carr-Price reaction product gave a spectral curve which was very different from those obtained from either e%gosterol or a crude commercial sample of 7-dehydrocholesterol.
FLUORESCENT
SUBSTANCES
IN
MARINE
BLOOD
WORM
141
be quite different as shown in Figs. 2-5 (er sample II) and in Fig. 6 (ez sample III). Moreover, none of these batches yielded the fluorescent compounds described earlier by Fox, Crane, and McConnaughey (1). Furthermore, samples II and III differed very widely from each other. The maximum extinction values for the numerous individual compounds were mostly observed in the region 220 to 280 rnp (in hexane). Figure 2 illustrates a special feature, viz. the occasional presence of aromatic polycyclic substances in the worms. The wavelength position of several (but not all) maxima of this fraction coincides with those typical for fluoranthene, a hydrocarbon which among others was recently identified in some barnacle extracts (4). E
400
300
v
FIG. 6. Extinction curves in hexane of fluorescent fractions and of carotenoid pigments (I-4) ez Z’horacophelia (sample III). (These fractions were separated chromatographically on calcium hydroxide-Celite 2:l by developing with hexane containing 04% acetone.)
Evidently, in accordance with the great variety of organic materials adsorbed to the ingest.ed sand, the marine blood worm is able to take up, and perhaps to store to a certain extent, fluorescent compounds of a wide chemical variety. In this connection there may be some significance to the demonstrated fact (1) that the worm not only fails to assimilate, but seems to degrade or otherwise alter the copious xanthophylls which greatly exceed the quantities of carotenes in its natural diet. SUMMARY
Extracts of two large samples (10-11 kg.) of the marine annelid worm Thoracophelia mwonata were examined, by means of chromatographic
142
FOX,
KOE,
PETK4CEK
AND ZECHMEISTER
fractionation in ultraviolet light, for fluorescing substances. A great variety and variability of such compounds were found to be present in the worms, collected at different times from the same habitat.
1. Fox, D. L., CRANE, S. C., AND MC~ONNAUGHEY, B. H., J. IMarine Research 7, 567 (1948). 2. KOE, B. K., Ph. D. Thesis, California Institute of Technology, Pasadena, Calif., 1952. 3. PETRACEK, F. J., M.S. Thesis, California Institute of Technology, Pasadena, Calif., 1951. 4. ZECHMEISTER, L., AND KOE, B. K., Arch. Biochem. Biophys. 36, 1 (1952).