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Hexosamine content of marine biological adhesives
MICROCHEMICAL
17, 456-461
JOURNAL
Hexosamine
Content
ALAN Department
F.
( 1972)
of Marine
KRIVIS
of Chemistry,
AND
Biological
MICHAEL
University
D.
of Akron,
Adhesives
MARTZ
Akron,
Ohio 44325
INTRODUCTION
Several marine organisms secrete an adhesive which permits the attachment of the creature to an undersea surface. The tenacity with which this kind of adhesive cements the organism to a substrate is well known by those who have had to remove marine fouling from a ship bottom, from pilings, or from buoys. In addition, contrary to the application requirements of most other adhesives, these marine adhesives are applied under water, to a wet surface. For these and a number of other reasons, studies to elucidate the composition and structure of several of these adhesives have been undertaken. In particular, the adhesive secreted by animals such as the mussel and the barnacle have been of interest (24, 7). Preliminary reports on the composition of cured samples of this type of adhesive indicated the presence of a proteinaceous material, the amino acid content of which differed somewhat according to species and/or report (3, 4, 7). However, a marked difference appeared in these reports concerning other possible components of this kind of adhesive. Specifically, the presence or absence of one or more hexoses or hexosamines is a major point of difference. For example, it was proposed that the adhesive was a polysaccharide which did not contain any nitrogen or nitrogenous components (2), or at another extreme, the presence of carbohydrates could not be demonstrated with certainty (7). In between these extremes, the presence of one hexosamine was reported for the barnacle adhesive but none for the mussel adhesive (3), or the presence of more than one hexosamine was proposed (4). The present report describes the experimental data confirming the presence of two hexosamines, glucosamine and galactosamine, as components of the mussel byssal thread and to offer a possible explanation for the disparities between the various reports cited. EXPERIMENTAL
METHODS
Live specimens of MytiEus edulis were obtained from Marine Biological Laboratory, Woods Hole, MA, and transferred to a salt water 456 Copyright All rights
0 of
1972 by Academic Press, reproduction in any form
Inc. reserved.
MARINE
BIOLOGICAL
ADHESIVES
457
aquarium in our laboratories. After allowing the animals to equilibrate in our laboratories, samples of byssal threads were cut from these specimens. The threads were cleaned thoroughly by abrasion with a brush, dried, and cut into suitably sized pieces. Milligram amounts of dried byssal thread were hydrolyzed in a sealed tube in 4 M hydrochloric acid at 87°C. for 4 hrs. After removal of most of the remaining hydrochloric acid, a portion of the brownish residue was taken up in a minimum of distilled water and analyzed by means of a ligand exchange thin layer chromatographic procedure which was devised for the purpose of separating hexosamines (6). In essence, samples of hydrolysate were spotted on sheets of Mallinkrodt ChromAR 500 which had been impregnated with copper ion; the sheets were developed in an alcoholic ammonia solvent system. Spots were visualized with a Ninhydrin spray. Other samples of hydrolysate were transferred to a column of Dowex-50 ion exchange resin and eluted with 2M hydrochloric acid (I). Aliquots of eluate fractions which were expected to contain hexosamines were chromatographed using the previously mentioned TLC procedure. Further hydrolysate samples were analyzed by means of a TLC method which can separate amino acids from hexosamines (5) ; the latter method is not capable of separating the various hexosamines, however. RESULTS
AND
DISCUSSION
Figure 1 is a photograph of a thin layer chromatogram of an aliquot of hydrolyzed mussel byssal thread. In addition to several other materials, galactosamine (spot No. 1)) and glucosamine (spot No. 2)) can be detected. In order to minimize possible interferences and establish the presence of these hexosamines even more firmly another aliquot of hydrolysate was subjected to a prior separtion via an ion exchange column. Eluate fractions which were expected to contain the hexosamines subsequently were chromatographed by means of the ligand exchange TLC procedure. Figure 2 shows a photograph of one of these chromatograms. The presence of galactosamine and glucosamine again can be detected (spot nos. 1 and 2, respectively) in several fractions. Another chromatographic procedure was applied to add further confirmatory evidence for the presence of the hexosamines. A twodimensional TLC procedure which had been developed for amino acid separations was utilized on aliquots of crude hydrolysate. This particular procedure separates the hexosamines, as a group, from the amino acids.
458
KRIVIS AND MARTZ
FIG. 1. Thin layer chromatogram galactosamine; Spot 2 = glucosamine.
of byssal thread hydrolysate.
Spot 1 =
The method, however, does not separate the different hexosamines from each other; several amino sugars appear as one spot on the chromatogram. When this technique was used to chromatograph an aliquot of byssal thread hydrolysate, a spot corresponding to the hexosamines appeared. Another chromatogram of an aliquot of hydrolysate with added known glucosamine showed the same spot slightly enlarged in diameter. From these various and several results, we can conclude that both glucosamine and galactosamine are present as components of Mytilus byssal threads. We can now address ourselves to the differences between the reports cited previously. The most logical reason for the disparities in the literature was gleaned from several unsuccessful experiments carried out in our laboratories. It was noted that when other samples of adhesive were hydrolyzed in 4 M hydrochloric acid for 4 hr at 92’C, only glucosamine could be detected in the hydrolysate (Fig. 3). Furthermore, when samples of byssal thread were hydrolyzed in 4 M hydrochloric acid for ca. 8 hr at 9%lOO”C, the chromatograms showed tw hexosamine to be
MARINE
BIOLOGICAL ADHESIVES
459
FIG. 2. Thin layer chromatogram of ionexchange eluates of byssal Thread hydrolysate. Spot 1 = galactosamine; Spot 2 = glucosamine.
present. In other words, a 5” elevation in the hydrolysis temperature could result in loss of one hexosamine while a 10” rise could destroy both hexosamines. Behavior of the sort outlined is not totally unexpected in dealing with this class of biological material (8). However, in the reports cited (2, 3, 7), the hydrolytic conditions utilized were aimed at protein hydrolysis and ultimately amino acid content. This purpose requires rather vigorous conditions such as high temperatures and long times of reaction. From our experiments, these conditions would be most likely to degrade one or more of the hexosamines which are present. Therefore, it appears probable that the reported absence of hexosamines in these marine adhesives was caused by inadvertent degradation of the materials during hydrolysis. SUMMARY Myths edulis byssal threads have been shown to contain both glucosamine and galactosamine. Inadvertent degradation of the material probably is the reason adhesive; evifor the reported absence of these hexosamines from the Myth dence for this explanation has been found.
460
KRIVIS
AND
MARTZ
FIG. 3. Thin layer chromatogram of byssal thread hydrolyzed at elevated temperature. Spot 1 = galactosamine; Spot 2 = glucosamine. ACKNOWLEDGMENT
The authors thank the National Institute of Dental Research for support through contract PH-43-67-1172. REFERENCES I. Boas, N. F., Method for the determination of hexosamines in tissues. J. Biol. Chem. 204, 553-563 (1953). 2. Cardarelli, N., Barnacle cement as a dental restorative adhesive. Nat. Inst. Health Publ. No. 151, 1968. 3. Cooke, M., private communication. 4. Krivis, A, F., Adhesives from the sea. Proc. Sot. Paint Tech., 12th Annu. Symp., Cleveland, Sept., 1970. 5. Krivis, A. F., and Ong, C. C., Thin-layer chromatography of amino acids. Microchem J. 16, 391-394 (1971). 6. Martz, M. D., and Krivis, A. F., Thin layer chromatography of hexosamines on copper impregnated sheets. Anal. Chem. 43, 790-791 (1971). 7. Saroyan, J. R., Lindner, E., Dooley, C. A., Bleile, H. R., Key to second generation antifouling coatings. Abstr. 158th Meet. Amer. Chem. Sot., 62-82 New York, Sept., 1969.
MARINE
BIOLOGICAL
ADHESIVES
461
8. S;Go, R. G., Characterization of carbohydrate units of glycoproteins. In Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. 8, pp. 26-52. Academic Press, New York. 1966.
17, 456-461
JOURNAL
Hexosamine
Content
ALAN Department
F.
( 1972)
of Marine
KRIVIS
of Chemistry,
AND
Biological
MICHAEL
University
D.
of Akron,
Adhesives
MARTZ
Akron,
Ohio 44325
INTRODUCTION
Several marine organisms secrete an adhesive which permits the attachment of the creature to an undersea surface. The tenacity with which this kind of adhesive cements the organism to a substrate is well known by those who have had to remove marine fouling from a ship bottom, from pilings, or from buoys. In addition, contrary to the application requirements of most other adhesives, these marine adhesives are applied under water, to a wet surface. For these and a number of other reasons, studies to elucidate the composition and structure of several of these adhesives have been undertaken. In particular, the adhesive secreted by animals such as the mussel and the barnacle have been of interest (24, 7). Preliminary reports on the composition of cured samples of this type of adhesive indicated the presence of a proteinaceous material, the amino acid content of which differed somewhat according to species and/or report (3, 4, 7). However, a marked difference appeared in these reports concerning other possible components of this kind of adhesive. Specifically, the presence or absence of one or more hexoses or hexosamines is a major point of difference. For example, it was proposed that the adhesive was a polysaccharide which did not contain any nitrogen or nitrogenous components (2), or at another extreme, the presence of carbohydrates could not be demonstrated with certainty (7). In between these extremes, the presence of one hexosamine was reported for the barnacle adhesive but none for the mussel adhesive (3), or the presence of more than one hexosamine was proposed (4). The present report describes the experimental data confirming the presence of two hexosamines, glucosamine and galactosamine, as components of the mussel byssal thread and to offer a possible explanation for the disparities between the various reports cited. EXPERIMENTAL
METHODS
Live specimens of MytiEus edulis were obtained from Marine Biological Laboratory, Woods Hole, MA, and transferred to a salt water 456 Copyright All rights
0 of
1972 by Academic Press, reproduction in any form
Inc. reserved.
MARINE
BIOLOGICAL
ADHESIVES
457
aquarium in our laboratories. After allowing the animals to equilibrate in our laboratories, samples of byssal threads were cut from these specimens. The threads were cleaned thoroughly by abrasion with a brush, dried, and cut into suitably sized pieces. Milligram amounts of dried byssal thread were hydrolyzed in a sealed tube in 4 M hydrochloric acid at 87°C. for 4 hrs. After removal of most of the remaining hydrochloric acid, a portion of the brownish residue was taken up in a minimum of distilled water and analyzed by means of a ligand exchange thin layer chromatographic procedure which was devised for the purpose of separating hexosamines (6). In essence, samples of hydrolysate were spotted on sheets of Mallinkrodt ChromAR 500 which had been impregnated with copper ion; the sheets were developed in an alcoholic ammonia solvent system. Spots were visualized with a Ninhydrin spray. Other samples of hydrolysate were transferred to a column of Dowex-50 ion exchange resin and eluted with 2M hydrochloric acid (I). Aliquots of eluate fractions which were expected to contain hexosamines were chromatographed using the previously mentioned TLC procedure. Further hydrolysate samples were analyzed by means of a TLC method which can separate amino acids from hexosamines (5) ; the latter method is not capable of separating the various hexosamines, however. RESULTS
AND
DISCUSSION
Figure 1 is a photograph of a thin layer chromatogram of an aliquot of hydrolyzed mussel byssal thread. In addition to several other materials, galactosamine (spot No. 1)) and glucosamine (spot No. 2)) can be detected. In order to minimize possible interferences and establish the presence of these hexosamines even more firmly another aliquot of hydrolysate was subjected to a prior separtion via an ion exchange column. Eluate fractions which were expected to contain the hexosamines subsequently were chromatographed by means of the ligand exchange TLC procedure. Figure 2 shows a photograph of one of these chromatograms. The presence of galactosamine and glucosamine again can be detected (spot nos. 1 and 2, respectively) in several fractions. Another chromatographic procedure was applied to add further confirmatory evidence for the presence of the hexosamines. A twodimensional TLC procedure which had been developed for amino acid separations was utilized on aliquots of crude hydrolysate. This particular procedure separates the hexosamines, as a group, from the amino acids.
458
KRIVIS AND MARTZ
FIG. 1. Thin layer chromatogram galactosamine; Spot 2 = glucosamine.
of byssal thread hydrolysate.
Spot 1 =
The method, however, does not separate the different hexosamines from each other; several amino sugars appear as one spot on the chromatogram. When this technique was used to chromatograph an aliquot of byssal thread hydrolysate, a spot corresponding to the hexosamines appeared. Another chromatogram of an aliquot of hydrolysate with added known glucosamine showed the same spot slightly enlarged in diameter. From these various and several results, we can conclude that both glucosamine and galactosamine are present as components of Mytilus byssal threads. We can now address ourselves to the differences between the reports cited previously. The most logical reason for the disparities in the literature was gleaned from several unsuccessful experiments carried out in our laboratories. It was noted that when other samples of adhesive were hydrolyzed in 4 M hydrochloric acid for 4 hr at 92’C, only glucosamine could be detected in the hydrolysate (Fig. 3). Furthermore, when samples of byssal thread were hydrolyzed in 4 M hydrochloric acid for ca. 8 hr at 9%lOO”C, the chromatograms showed tw hexosamine to be
MARINE
BIOLOGICAL ADHESIVES
459
FIG. 2. Thin layer chromatogram of ionexchange eluates of byssal Thread hydrolysate. Spot 1 = galactosamine; Spot 2 = glucosamine.
present. In other words, a 5” elevation in the hydrolysis temperature could result in loss of one hexosamine while a 10” rise could destroy both hexosamines. Behavior of the sort outlined is not totally unexpected in dealing with this class of biological material (8). However, in the reports cited (2, 3, 7), the hydrolytic conditions utilized were aimed at protein hydrolysis and ultimately amino acid content. This purpose requires rather vigorous conditions such as high temperatures and long times of reaction. From our experiments, these conditions would be most likely to degrade one or more of the hexosamines which are present. Therefore, it appears probable that the reported absence of hexosamines in these marine adhesives was caused by inadvertent degradation of the materials during hydrolysis. SUMMARY Myths edulis byssal threads have been shown to contain both glucosamine and galactosamine. Inadvertent degradation of the material probably is the reason adhesive; evifor the reported absence of these hexosamines from the Myth dence for this explanation has been found.
460
KRIVIS
AND
MARTZ
FIG. 3. Thin layer chromatogram of byssal thread hydrolyzed at elevated temperature. Spot 1 = galactosamine; Spot 2 = glucosamine. ACKNOWLEDGMENT
The authors thank the National Institute of Dental Research for support through contract PH-43-67-1172. REFERENCES I. Boas, N. F., Method for the determination of hexosamines in tissues. J. Biol. Chem. 204, 553-563 (1953). 2. Cardarelli, N., Barnacle cement as a dental restorative adhesive. Nat. Inst. Health Publ. No. 151, 1968. 3. Cooke, M., private communication. 4. Krivis, A, F., Adhesives from the sea. Proc. Sot. Paint Tech., 12th Annu. Symp., Cleveland, Sept., 1970. 5. Krivis, A. F., and Ong, C. C., Thin-layer chromatography of amino acids. Microchem J. 16, 391-394 (1971). 6. Martz, M. D., and Krivis, A. F., Thin layer chromatography of hexosamines on copper impregnated sheets. Anal. Chem. 43, 790-791 (1971). 7. Saroyan, J. R., Lindner, E., Dooley, C. A., Bleile, H. R., Key to second generation antifouling coatings. Abstr. 158th Meet. Amer. Chem. Sot., 62-82 New York, Sept., 1969.
MARINE
BIOLOGICAL
ADHESIVES
461
8. S;Go, R. G., Characterization of carbohydrate units of glycoproteins. In Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. 8, pp. 26-52. Academic Press, New York. 1966.