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Comparative studies of mucopolysaccharides from marine animals. II. Illex illecebrosvs (Lesueur) and various crustaceans
J. exp. mar. &of. Ecof., 1981, Vol. 52, pp. 95-101
Elsevier/North-Holland
95
Biomedical Press
COMPARATIVE
STUDIES
MARINE ANIMALS.
OF MUCOPOLYSACCHARIDES
II. ZLLEX ZLLECEBZZOSUS
VARIOUS
FROM
(Lesueur) AND
CRUSTACEANS
J. EHRLICH College of Medicine and Dentistry of New Jersey, Newark, NJ 07103, U.S.A.
B. PATEL’ and S. S. STIVALA Department of Chemistry and Chemical engineering, Stevens ~~tit~te of Technology, ~oboken~ NJ 0703t3, U.S.A.
Abstract: Various mucopolysaccharides (MPS) were extracted from the tentacles of the squid, I&x iliecebrosus (Leseuer) and the viscera of crustaceans comprising the blue crab, Cailinectes sapidus Rathbun, the green crab, Carcinus maenas (Linne), the red crab, Geryon quinquedens Smith, the rock crab, Cancer irroratus Say, the lobster (body and head), Homarus americana Milne Edwards, and the shrimp (head), Pandalus borealis (Kroyer). The MPS were analyzed for uranic acid, hexosamine, N-sulfate, protein, neutral sugar, and anticoagulant activity. Chemical analysis of the two fractions extracted from the squid tentacles, suggests that fraction Fl is similar to chondroitin sulfate and F2 is heparin-like. In the crustaceans, the MPS extracted appear to resemble chondroitin sulfate and heparin. The blood anticoagulant activity of the MPS from the red crab was ~66 iU/mg, whereas those obtained from the other species ranged from 7 to 30 IU/mg. Based on these data and the chemical analysis, it appears that the MPS from the red crab is heparin-like, while the MPS from the other species are more like chondroitin sulfate.
The literature contains a good deal of information on the mucopolysaccharides (MPS) derived from vertebrates, particularly the mammals. Although much less has been reported on the MPS derived from invertebrates, there is, however, evidence of their presence in some of these organisms. Heparin-like MPS have been isolated from the clams, Spisula solidissima and Artica islandica, called Mactins A and B, respectively, by Frommhagen et al. (1953) and Burson et al. (1956); these MPS exhibit high anticoagulant activities. Various types of chondroitin sulfates have been located in the squid, e.g., Mathews et al. (1962) found over-sulfated chondroitin sulfate in the cartilage of the squid, Lo&go, Srinivasan et al. (1969) located chondroitin and highly sulfated chondroitin sulfate in the skin t Present address: Roy Weston. Inc., West Chester, PA 19380, U.S.A. 0022-0981/81/00~-0~0/$2.50
0 Elsevier~orth-Holland
Biomedical Press
96
J.EHRLtCHETAL.
of the squid, ~~Iigo opa~escens, and the skin of ~mmastrep~es sloani pa~~~~~ yielded a non-sulfated chondroitin (Anno et al., 1964; Suzuki et al., 4968). The presence of heparin-like compounds (in addition to other MPS) in six molluscans, including the L&go brasiliense, was reported by Cassaro & Dietrich (1977). The sulfated MPS from Lo&go were found to contain a chondroitin sulfate similar to the chondroitin sulfate-E extracted from the cartilaginous tissue of the squid by Kawai et al. (1966) and Suzuki et al. (1968). In crustaceans, Mathews et al. (1962) reported an oversulfated chondroitin sulfate from the horseshoe crab, Limuhs, Seno et al. (1974) extracted chondroitin sulfate-K from the cartilage of the king crab, Tachypleus tridentatus, and Cassaro 8z Dietrich (1977) reported that the sulfated MPS from some shrimp and crabs were heparitin sulfates, whereas lobsters contained a heparin-like compound. This paper is a continuation of earlier work conducted in this laboratory on the comparative studies of MPS from marine animals. In this connection, Pate1 et al. (1980) reported on the MPS extracted from the skate, Baja eg~anteria. The present study was undertaken to extract and characterize the MPS from tissues of the squid, Illex illecebrosus (Leseuer), the lobster, Homarus americana Milne Edwards, the blue crab, Callinectes sapidus Rathbun, the green crab, Carcinus maenas (Lint&), the red crab, Geryon guinquedens Smith, the rock crab, Cancer irroratus Say, and the shrimp, Pandalus borealis (Kriiyer).
MATERIALANDMETHODS
The animals were obtained from the National Marine Fisheries Service, Gloucester, Mass. The tentacles of the squid, the viscera of the crab, the head and body of the lobster and the head of the shrimp were employed in obtaining the MPS for this study. The MPS were extracted from the defatted parts according to the procedures described by Pate1 et al. (1980). In the case of the squid and blue crab, two fractions were obtained on elution from the anion-exchange column with 1 M and 2 M NaCl, which will be identified as fraction Fl and fraction FZ, respectively. All of the other samples consisted mainly of the 2 M eluant. The electrophoresis, chemical analysis and anticoagulant tests of the various MPS were performed according to the procedures described by Pate1 et al. (1980). RESULTSAND
DISCUSSION
Preliminary identification of the MPS extracted from the various samples were made by observing the electrophoretic mobilities on agarose gel in acetate buffer at pH 3. These observations were supported by additional data from chemical analyses. Figs. 1 and 2 show el~trophoreto~ams of the extracted MPS. Table I summarizes the correspon~ng chemical analysis. For purposes of ~ompa~son, the
MUCOPOLYSACCHARIDES
FROM
MARINE
ANIMALS
97
figures and table also contain standard heparin, chondroitin sulfate, keratosulfate, and heparitin sulfate. It should be mentioned that the C/O ratio (carbazole/orcinol) is a measure of the ratio of glucuronic/iduronic acid concentrations. SQUID
It is noted from Table I that the uranic acid value of 23.7% of Fl is in close agreement to 24.2% of chondroitin sulfate. The C/O ratio is 1.15 compared to 1.42 of chondroitin sulfate. This ratio is lower than that of the MPS extracted from the skin of the squid by Anno et al. (1964). They reported the value of 1.64, from Ommastrephes sloani pacificus, indicating that the MPS had a higher glucuronic acid content than iduronic acid. The percentage of hexosamine and the H/C ratio (hexosamine/carbazole) of Fl and F2 fractions are closer to those of heparin than to those of chondroitin sulfate. Furthermore, these values are in reasonably good agreement with the value (0.787) reported by Cassaro & Dietrich (1977) for the MPS extracted from the squid, Lolligo brasiliense (it is of interest to note that the entire organism was used in this case). On the other hand, the H/C value of 1.02 was reported by Anno et al. (1964) on the MPS obtained from the skin of Omnastrephes sloani pacificus, but this MPS was non-sulfated and was identified as chondroitin. From the total sulfate analysis, it can be seen that the Fl fraction is a less highly sulfated MPS than heparin. The F2 fraction, on the other hand, is more sulfated and thus more closely resembles heparin. The value of 1.41 for the S/H ratio (total sulfate/hexosamine) is of the same order of magnitude for the Fl fraction as that obtained by Cassaro & Dietrich (1977) for Lolligo brasiliense (1.6). Furthermore, the value for this fraction is closer to that of chondroitin sulfate, whereas the value of 3.31 for the F2 fraction is closer to that of heparin. The anticoagulant activities of the Fl and F2 fractions are considerably lower than that of heparin. The electrophoretogram of the F2 fraction suggests two components, one with a migration closer to heparin and the other closer to chondroitin sulfate. On the basis of the chemical analyses, it appears that the MPS extracted from the tentacles of Illex illecebrosus may contain both chondroitin sulfate and heparin-like components; the Fl fraction is, however, closer in composition to chondroitin sulfate and the F2 fraction is closer to heparin. CRUSTACEANS
The electrophoresis pattern of the MPS isolated from the various crab viscera is shown in Fig. 1, and those derived from the lobster head and body, and shrimp head are shown in Fig. 2. It may be noted that the MPS from the red crab, lobster head and body appear to contain some heparin-like components, whereas the rock, green, and blue crabs show varying amounts of chondroitin sulfate, but no heparitin sulfate or keratosulfate. The shrimp head material has a similar mobility to
I
35.0 .21.0 0.650 31.1 3.31 5.33 25.8 -
27
27.8 23.7 1.17 19.2 0.748 12.1 1.41 1.15 10.9 1.41 4.20
21 64-68
33.6 17.2 1.95 18.9 0.610 31.1 3.68 12.8 18.3 1.55 3.38
Red crab
7-10
11.2 15.4 0.727 18.6 1.80 10.2 1.23 2.46 7.74 0.290 10.8
Fl
lo-12
13.3 18.0 0.739 19.6 1.60 11.0 1.26 5.39 5.61 0.718 13.8
F2
Blue crab
~-
10-14
22.6 19.8 1.14 17.1 0.820 17.1 2.24 6.7 10.4 0.626 10.8
Rock crab
18-21
37.2 17.4 2.14 16.8 0.489 25.5 3.40 8.31 17.2 0.143 22.0
Green crab
28-30
12.0 2.58 4.65 5.61 0.507 7.67 1.67 2.96
head
28-30
16.6 18.6 0.892 10.9 0.712 19.1 3.92 6.70 12.4 4.85 10.3
body
Lobster
a Analysis performed by authors on CS obtained from ICN Pharmaceuticals, Inc., reported to be 99%. b Data for KS and HS taken from Roden et al. (1972). ’ Obtained by subtraction of N-sulfate from total sulfate
--.. Uranic acid Carbazole (C) Orcinol (0) C/G Hexosamine (H) WC Total sulfate (S) S/H N-sulfate 0-sulfatec Protein Neutral sugar Anticoagulant activity IU/mg
Squid tentacles --Fl F2
22
23.3 13.6 1.71 11.3 0.526 17.6 3.49 1.90 15.7 6.42 19.2
Shrimp head
164
32.3 12.7 2.54 20.3 0.681 23.7 2.61 13.5 10.2 0.799 3.67
Heparin
-
34.3 24.2 1.42 26.5 0.837 13.5 1.14 0 13.5 1.81 5.39
CSa
_
1.9 29.3 17 15 1.17 Trace 36.9
KSb
44.1 24.6 0.604 11 0.99 Trace -
HSb
The chemical analyses of mucopolysaccha~des from the squid and crustaceans: values are reported in “/, by weight, however, ratios indicated in the table are molar ratios; CS, chon~oitin sulfate; KS, keratosutfate; HS,heparitin sulfate.
TABLE
Fig. crab
1. Electrophoretic viscera
MPS,
patterns on agarose gel: reading from left to right, red crab viscera MPS, rock blue crab viscera MPS, green crab viscera MPS, heparin, chondroitin sulfate. heparitin sulfate, keratosulfate.
Fig. 2. Electrophoretic patterns on agarose gel: reading body MPS. shrimp head MPS, heparin, chondroitin
99
from left to right, lobster head MPS, sulfate, heparitin sulfate. keratosulfate.
lobster
100
J. EHRLICH ET AL
chondroitin sulfate and one component of heparin, but that of the lobster body is similar to chondroitin sulfate, heparin, heparitin sulfate or keratosulfate. The chemical analysis data of the crustacean samples are shown in Table I. The carbazole assay results suggest that the red and green crab MPS are closely similar to either heparin or chondroitin sulfate in uranic acid composition; however, the orcinol analysis indicates a closer relationship with heparin rather than with chondroitin sulfate for red and green crab, and shrimp head material. The C/O ratio is higher for the red and green crab material, and lobster head, suggesting that there is more glucuronic than iduronic acid in the MPS of these species. A value of similar magnitude (1.89) was found for the gill cartilage of the king crab, Tachypleus tridentatus (Seno et al., 1974). Heparin likewise gives a high value (2.54). The percentages of hexosamine in all the crab viscera examined are all reasonably close to that of heparin, while those of lobster head and body, and shrimp head are considerably lower in value. It may be noted that the molar ratios of hexosamine to uranic acid found in the red crab MPS fit more closely to heparin or heparitin sulfate, while the results for rock crab material are similar to those for chondroitin sulfate. The value of this ratio for whole blue crab obtained by Cassaro & Dietrich (1977) was 0.625, whereas the values of 1.80 and 1.60 were obtained for our viscera fractions Fl and F2, respectively. It may also be noted that a ratio of slightly > 1 was reported for the king crab (2M fraction) by Seno et a/. (1974). From the percentages of total sulfate, it would appear that the MPS from red and green crabs, and lobster body are closer in composition to heparin, while those from blue crab (Fl and F2) are closer to heparitin or chondroitin sulfate. The N-sulfate value for red crab material approaches the value for heparin, and that of shrimp head is closer to that for chondroitin sulfate. The S/H ratios of all crustacean samples are > 1, suggesting that they all contain fairly highly sulfated MPS. This ratio in the king crab was found to be 1.64 by Seno et al. (1974), in the horseshoe crab 1.45 by Mathews et al. (1962) and in the blue crab 1.57 by Cassaro & Dietrich (1977). The latter value compares fairly closely to our results for the blue crab. The amount of neutral sugars present in the red crab and lobster MPS is similar to that in heparin. The anti-coagulant activities of all the crustacean samples, with the exception of the red crab, are low. This may be due to the higher neutral sugar content as well as to a lower heparin-like composition of those samples exhibiting the lower activities. Naturally occurring chondroitin sulfate has no heparin-like anticoagulant activity (Nagasawa et al., 1972). In summary, blue crab (FI and F2) MPS are quite similar in their chemical analysis. According to their electrophoretic mobilities and total sulfate content, the blue crab fractions resemble chondroitin sulfate. Because of the fairly high percentage of sulfate in the lobster body sample, and the large band in the electrophoretogram, it might be concluded that some heparinoid is present, as well as smaller amounts of some of the other MPS. The shrimp head MPS component resembles chondroitin sulfate in terms of its low N-sulfate, but from its anti-
MUCOPOLYSAC~HARIDES
FROM MARINE ANIMALS
101
coagulant activity, could have some heparin-like compound as well. The rock crab MPS, from its total sulfate analysis and low anticoagulant activity, appears to be mostly composed of chondroitin sulfate. The green crab MPS, from its high total and N-sulfate composition, and its electrophoretic mobility, contains some heparinlike component. The red crab MPS, from its high anticoagulant activity and its similarity to heparin in chemical analysis, as well as its similarity in electrophoresis, appears to have a heparin-like compound as its major component. It is also interesting to note that all the samples examined in this study, from their S/H ratios contain MPS that are over-sulfated, such as was found in the skate, Ruja eglanteria (Pate1 et al., 1980). ACKNOWLEDGEMENTS
This work is a result of research sponsored, in part, by NOAA, Office of Sea Grant, Department of Commerce to Stevens Institute of Technology, under Grant Number 04-7-158-44042. The authors are grateful to Mr. R. Leason and Ms. M. Perry, National Fisheries, NOAA, Gloucester, Mass. for samples and technical assistance.
REFERENCES
ANNO, K., Y.
KAWAI &
N. SENO, 1964. Isolation of chondroitin
from squid skin. ~jochim.
biophys.
Acta, Vol. 83, pp. 348-349.
M. J. FAHRENBACFI, L. H. FROMMHAGEN,B.A. RICCARDI,R.A. BROWN, J.A. BROCKLEWRY& E.L.R. STOVSTAD, 1956. Isolation and purification of mactins, heparin-like anticoagulants from Moiiusca. J. Am. them. Sot., Vol. 78, pp. 5874-5878. CASSARO,C.M.F. & C.P. DIETRICH, 1977. Distribution of sulfated mucopolysaccharides in invertebrates, J. biol. Chem., Vol. 52, pp. 2254-2261. FROMMHAGEN,L. H., M. J. FAHRENBACH,J. A. BROCKMAN& E. L. R. STOVSTAD,1953. Heparin-like anticoagulants from Moiiusca. Proc. Sac. exp. Biol. Med., Vol. 82, pp. 28&283. KAWAI, Y., N. SENO & K. ANNO, 1966. Chondroitin polysulfate of squid cartilage. J. Biochem, Tokyo, Vol. 60, pp. 317-321. MATHEWS, M.B., J. DUH & P. PERSON, 1962. Acid mucopoiysaccharides of invertebrate cartilage. Nature, Land., Vol. 193, pp. 378-379. NAGASAWA,K.A. OGAMO & S. HAYASHI, 1972. Heparinoid activities of depoiyme~zed poiysuifates. Archs Biochem. Biophys., Vol. 158, pp. 451-458. PATEL,B., J. EHRLICH,S. S. STIVALA& N. K. SINGH,1980. Comparative studies of mucopoiysaccharides from marine animals. I. Rajo eglanteria Bose. J. exp. mar. Biol. Erol., Vol. 46, pp. 127-135. RODEN, L., J. F. BAKER, A. CIFONELLI& M. B. MATHEWS,1972. Isolation and characterization of connective tissue polysaccharides. In, Methods in enzymology, Vol. 28, edited by V. Ginsburg, Academic Press, New York, pp. 73-140. SENO, N., S. YAMASHIRO& K. ANNO, 1974. Isolation and characterization of a new disaccaride disulfate: 2-acetoamido-2-deoxy-3-O-(2or 3-O-suifo+D-giucopyranosyiuronic acid)-4-O-suifo-Dgaiactose. Biochim. biophys. Acta, Vol. 343, pp. 423-426. SRINIVASAN, S. R., B. RADHAKRISHNAMURTHY, E. R. DALFERES& G. S. BERENSON,1969. Glycosaminoglycans from squid skin. Comp. Biochem. Physiol., Vol. 28, pp. 169-176. SUZUKI,S., H. SAITO, T. YAMAGATA,K. ANNO, N. SENO, Y. KAWAI&T. FURUHASHI,1968. Formation of three types of &sulfated disaccharides from chondroitin sulfates by chondroitinase digestion. J. biol. Chem., Vol. 243, pp. 1543-i 550. BURSON,SK., MAN, H.V.
Elsevier/North-Holland
95
Biomedical Press
COMPARATIVE
STUDIES
MARINE ANIMALS.
OF MUCOPOLYSACCHARIDES
II. ZLLEX ZLLECEBZZOSUS
VARIOUS
FROM
(Lesueur) AND
CRUSTACEANS
J. EHRLICH College of Medicine and Dentistry of New Jersey, Newark, NJ 07103, U.S.A.
B. PATEL’ and S. S. STIVALA Department of Chemistry and Chemical engineering, Stevens ~~tit~te of Technology, ~oboken~ NJ 0703t3, U.S.A.
Abstract: Various mucopolysaccharides (MPS) were extracted from the tentacles of the squid, I&x iliecebrosus (Leseuer) and the viscera of crustaceans comprising the blue crab, Cailinectes sapidus Rathbun, the green crab, Carcinus maenas (Linne), the red crab, Geryon quinquedens Smith, the rock crab, Cancer irroratus Say, the lobster (body and head), Homarus americana Milne Edwards, and the shrimp (head), Pandalus borealis (Kroyer). The MPS were analyzed for uranic acid, hexosamine, N-sulfate, protein, neutral sugar, and anticoagulant activity. Chemical analysis of the two fractions extracted from the squid tentacles, suggests that fraction Fl is similar to chondroitin sulfate and F2 is heparin-like. In the crustaceans, the MPS extracted appear to resemble chondroitin sulfate and heparin. The blood anticoagulant activity of the MPS from the red crab was ~66 iU/mg, whereas those obtained from the other species ranged from 7 to 30 IU/mg. Based on these data and the chemical analysis, it appears that the MPS from the red crab is heparin-like, while the MPS from the other species are more like chondroitin sulfate.
The literature contains a good deal of information on the mucopolysaccharides (MPS) derived from vertebrates, particularly the mammals. Although much less has been reported on the MPS derived from invertebrates, there is, however, evidence of their presence in some of these organisms. Heparin-like MPS have been isolated from the clams, Spisula solidissima and Artica islandica, called Mactins A and B, respectively, by Frommhagen et al. (1953) and Burson et al. (1956); these MPS exhibit high anticoagulant activities. Various types of chondroitin sulfates have been located in the squid, e.g., Mathews et al. (1962) found over-sulfated chondroitin sulfate in the cartilage of the squid, Lo&go, Srinivasan et al. (1969) located chondroitin and highly sulfated chondroitin sulfate in the skin t Present address: Roy Weston. Inc., West Chester, PA 19380, U.S.A. 0022-0981/81/00~-0~0/$2.50
0 Elsevier~orth-Holland
Biomedical Press
96
J.EHRLtCHETAL.
of the squid, ~~Iigo opa~escens, and the skin of ~mmastrep~es sloani pa~~~~~ yielded a non-sulfated chondroitin (Anno et al., 1964; Suzuki et al., 4968). The presence of heparin-like compounds (in addition to other MPS) in six molluscans, including the L&go brasiliense, was reported by Cassaro & Dietrich (1977). The sulfated MPS from Lo&go were found to contain a chondroitin sulfate similar to the chondroitin sulfate-E extracted from the cartilaginous tissue of the squid by Kawai et al. (1966) and Suzuki et al. (1968). In crustaceans, Mathews et al. (1962) reported an oversulfated chondroitin sulfate from the horseshoe crab, Limuhs, Seno et al. (1974) extracted chondroitin sulfate-K from the cartilage of the king crab, Tachypleus tridentatus, and Cassaro 8z Dietrich (1977) reported that the sulfated MPS from some shrimp and crabs were heparitin sulfates, whereas lobsters contained a heparin-like compound. This paper is a continuation of earlier work conducted in this laboratory on the comparative studies of MPS from marine animals. In this connection, Pate1 et al. (1980) reported on the MPS extracted from the skate, Baja eg~anteria. The present study was undertaken to extract and characterize the MPS from tissues of the squid, Illex illecebrosus (Leseuer), the lobster, Homarus americana Milne Edwards, the blue crab, Callinectes sapidus Rathbun, the green crab, Carcinus maenas (Lint&), the red crab, Geryon guinquedens Smith, the rock crab, Cancer irroratus Say, and the shrimp, Pandalus borealis (Kriiyer).
MATERIALANDMETHODS
The animals were obtained from the National Marine Fisheries Service, Gloucester, Mass. The tentacles of the squid, the viscera of the crab, the head and body of the lobster and the head of the shrimp were employed in obtaining the MPS for this study. The MPS were extracted from the defatted parts according to the procedures described by Pate1 et al. (1980). In the case of the squid and blue crab, two fractions were obtained on elution from the anion-exchange column with 1 M and 2 M NaCl, which will be identified as fraction Fl and fraction FZ, respectively. All of the other samples consisted mainly of the 2 M eluant. The electrophoresis, chemical analysis and anticoagulant tests of the various MPS were performed according to the procedures described by Pate1 et al. (1980). RESULTSAND
DISCUSSION
Preliminary identification of the MPS extracted from the various samples were made by observing the electrophoretic mobilities on agarose gel in acetate buffer at pH 3. These observations were supported by additional data from chemical analyses. Figs. 1 and 2 show el~trophoreto~ams of the extracted MPS. Table I summarizes the correspon~ng chemical analysis. For purposes of ~ompa~son, the
MUCOPOLYSACCHARIDES
FROM
MARINE
ANIMALS
97
figures and table also contain standard heparin, chondroitin sulfate, keratosulfate, and heparitin sulfate. It should be mentioned that the C/O ratio (carbazole/orcinol) is a measure of the ratio of glucuronic/iduronic acid concentrations. SQUID
It is noted from Table I that the uranic acid value of 23.7% of Fl is in close agreement to 24.2% of chondroitin sulfate. The C/O ratio is 1.15 compared to 1.42 of chondroitin sulfate. This ratio is lower than that of the MPS extracted from the skin of the squid by Anno et al. (1964). They reported the value of 1.64, from Ommastrephes sloani pacificus, indicating that the MPS had a higher glucuronic acid content than iduronic acid. The percentage of hexosamine and the H/C ratio (hexosamine/carbazole) of Fl and F2 fractions are closer to those of heparin than to those of chondroitin sulfate. Furthermore, these values are in reasonably good agreement with the value (0.787) reported by Cassaro & Dietrich (1977) for the MPS extracted from the squid, Lolligo brasiliense (it is of interest to note that the entire organism was used in this case). On the other hand, the H/C value of 1.02 was reported by Anno et al. (1964) on the MPS obtained from the skin of Omnastrephes sloani pacificus, but this MPS was non-sulfated and was identified as chondroitin. From the total sulfate analysis, it can be seen that the Fl fraction is a less highly sulfated MPS than heparin. The F2 fraction, on the other hand, is more sulfated and thus more closely resembles heparin. The value of 1.41 for the S/H ratio (total sulfate/hexosamine) is of the same order of magnitude for the Fl fraction as that obtained by Cassaro & Dietrich (1977) for Lolligo brasiliense (1.6). Furthermore, the value for this fraction is closer to that of chondroitin sulfate, whereas the value of 3.31 for the F2 fraction is closer to that of heparin. The anticoagulant activities of the Fl and F2 fractions are considerably lower than that of heparin. The electrophoretogram of the F2 fraction suggests two components, one with a migration closer to heparin and the other closer to chondroitin sulfate. On the basis of the chemical analyses, it appears that the MPS extracted from the tentacles of Illex illecebrosus may contain both chondroitin sulfate and heparin-like components; the Fl fraction is, however, closer in composition to chondroitin sulfate and the F2 fraction is closer to heparin. CRUSTACEANS
The electrophoresis pattern of the MPS isolated from the various crab viscera is shown in Fig. 1, and those derived from the lobster head and body, and shrimp head are shown in Fig. 2. It may be noted that the MPS from the red crab, lobster head and body appear to contain some heparin-like components, whereas the rock, green, and blue crabs show varying amounts of chondroitin sulfate, but no heparitin sulfate or keratosulfate. The shrimp head material has a similar mobility to
I
35.0 .21.0 0.650 31.1 3.31 5.33 25.8 -
27
27.8 23.7 1.17 19.2 0.748 12.1 1.41 1.15 10.9 1.41 4.20
21 64-68
33.6 17.2 1.95 18.9 0.610 31.1 3.68 12.8 18.3 1.55 3.38
Red crab
7-10
11.2 15.4 0.727 18.6 1.80 10.2 1.23 2.46 7.74 0.290 10.8
Fl
lo-12
13.3 18.0 0.739 19.6 1.60 11.0 1.26 5.39 5.61 0.718 13.8
F2
Blue crab
~-
10-14
22.6 19.8 1.14 17.1 0.820 17.1 2.24 6.7 10.4 0.626 10.8
Rock crab
18-21
37.2 17.4 2.14 16.8 0.489 25.5 3.40 8.31 17.2 0.143 22.0
Green crab
28-30
12.0 2.58 4.65 5.61 0.507 7.67 1.67 2.96
head
28-30
16.6 18.6 0.892 10.9 0.712 19.1 3.92 6.70 12.4 4.85 10.3
body
Lobster
a Analysis performed by authors on CS obtained from ICN Pharmaceuticals, Inc., reported to be 99%. b Data for KS and HS taken from Roden et al. (1972). ’ Obtained by subtraction of N-sulfate from total sulfate
--.. Uranic acid Carbazole (C) Orcinol (0) C/G Hexosamine (H) WC Total sulfate (S) S/H N-sulfate 0-sulfatec Protein Neutral sugar Anticoagulant activity IU/mg
Squid tentacles --Fl F2
22
23.3 13.6 1.71 11.3 0.526 17.6 3.49 1.90 15.7 6.42 19.2
Shrimp head
164
32.3 12.7 2.54 20.3 0.681 23.7 2.61 13.5 10.2 0.799 3.67
Heparin
-
34.3 24.2 1.42 26.5 0.837 13.5 1.14 0 13.5 1.81 5.39
CSa
_
1.9 29.3 17 15 1.17 Trace 36.9
KSb
44.1 24.6 0.604 11 0.99 Trace -
HSb
The chemical analyses of mucopolysaccha~des from the squid and crustaceans: values are reported in “/, by weight, however, ratios indicated in the table are molar ratios; CS, chon~oitin sulfate; KS, keratosutfate; HS,heparitin sulfate.
TABLE
Fig. crab
1. Electrophoretic viscera
MPS,
patterns on agarose gel: reading from left to right, red crab viscera MPS, rock blue crab viscera MPS, green crab viscera MPS, heparin, chondroitin sulfate. heparitin sulfate, keratosulfate.
Fig. 2. Electrophoretic patterns on agarose gel: reading body MPS. shrimp head MPS, heparin, chondroitin
99
from left to right, lobster head MPS, sulfate, heparitin sulfate. keratosulfate.
lobster
100
J. EHRLICH ET AL
chondroitin sulfate and one component of heparin, but that of the lobster body is similar to chondroitin sulfate, heparin, heparitin sulfate or keratosulfate. The chemical analysis data of the crustacean samples are shown in Table I. The carbazole assay results suggest that the red and green crab MPS are closely similar to either heparin or chondroitin sulfate in uranic acid composition; however, the orcinol analysis indicates a closer relationship with heparin rather than with chondroitin sulfate for red and green crab, and shrimp head material. The C/O ratio is higher for the red and green crab material, and lobster head, suggesting that there is more glucuronic than iduronic acid in the MPS of these species. A value of similar magnitude (1.89) was found for the gill cartilage of the king crab, Tachypleus tridentatus (Seno et al., 1974). Heparin likewise gives a high value (2.54). The percentages of hexosamine in all the crab viscera examined are all reasonably close to that of heparin, while those of lobster head and body, and shrimp head are considerably lower in value. It may be noted that the molar ratios of hexosamine to uranic acid found in the red crab MPS fit more closely to heparin or heparitin sulfate, while the results for rock crab material are similar to those for chondroitin sulfate. The value of this ratio for whole blue crab obtained by Cassaro & Dietrich (1977) was 0.625, whereas the values of 1.80 and 1.60 were obtained for our viscera fractions Fl and F2, respectively. It may also be noted that a ratio of slightly > 1 was reported for the king crab (2M fraction) by Seno et a/. (1974). From the percentages of total sulfate, it would appear that the MPS from red and green crabs, and lobster body are closer in composition to heparin, while those from blue crab (Fl and F2) are closer to heparitin or chondroitin sulfate. The N-sulfate value for red crab material approaches the value for heparin, and that of shrimp head is closer to that for chondroitin sulfate. The S/H ratios of all crustacean samples are > 1, suggesting that they all contain fairly highly sulfated MPS. This ratio in the king crab was found to be 1.64 by Seno et al. (1974), in the horseshoe crab 1.45 by Mathews et al. (1962) and in the blue crab 1.57 by Cassaro & Dietrich (1977). The latter value compares fairly closely to our results for the blue crab. The amount of neutral sugars present in the red crab and lobster MPS is similar to that in heparin. The anti-coagulant activities of all the crustacean samples, with the exception of the red crab, are low. This may be due to the higher neutral sugar content as well as to a lower heparin-like composition of those samples exhibiting the lower activities. Naturally occurring chondroitin sulfate has no heparin-like anticoagulant activity (Nagasawa et al., 1972). In summary, blue crab (FI and F2) MPS are quite similar in their chemical analysis. According to their electrophoretic mobilities and total sulfate content, the blue crab fractions resemble chondroitin sulfate. Because of the fairly high percentage of sulfate in the lobster body sample, and the large band in the electrophoretogram, it might be concluded that some heparinoid is present, as well as smaller amounts of some of the other MPS. The shrimp head MPS component resembles chondroitin sulfate in terms of its low N-sulfate, but from its anti-
MUCOPOLYSAC~HARIDES
FROM MARINE ANIMALS
101
coagulant activity, could have some heparin-like compound as well. The rock crab MPS, from its total sulfate analysis and low anticoagulant activity, appears to be mostly composed of chondroitin sulfate. The green crab MPS, from its high total and N-sulfate composition, and its electrophoretic mobility, contains some heparinlike component. The red crab MPS, from its high anticoagulant activity and its similarity to heparin in chemical analysis, as well as its similarity in electrophoresis, appears to have a heparin-like compound as its major component. It is also interesting to note that all the samples examined in this study, from their S/H ratios contain MPS that are over-sulfated, such as was found in the skate, Ruja eglanteria (Pate1 et al., 1980). ACKNOWLEDGEMENTS
This work is a result of research sponsored, in part, by NOAA, Office of Sea Grant, Department of Commerce to Stevens Institute of Technology, under Grant Number 04-7-158-44042. The authors are grateful to Mr. R. Leason and Ms. M. Perry, National Fisheries, NOAA, Gloucester, Mass. for samples and technical assistance.
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