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Isolation and amino acid composition of the collagen of white shrimp (Penaeus setiferous)—I
Comp. Biochem. Physiol., 1968, Voi. 27, pp. 127 to 132. PergamonPress. Printed in Great Britain
ISOLATION AND AMINO ACID COMPOSITION OF THE COLLAGEN OF WHITE SHRIMP
(PENAEUS SETIFEROUS)--I H A R O L D C. T H O M P S O N , JR. and MARY H. T H O M P S O N Teclmological Laboratory, Bureau of Commercial Fisheries, Pascegoula, Mis,,itmippi 39567, U.S.A. (Received 22 March 1968)
Atmtract--1. The methodology employed in isolating the insoluble collagen of white shrimp (Penaeus setiferous) is reported. 2. The physical and chemical characteristics of the isolated protein are discussed including amino acid composition, total nitrogen content, hexose content, amide nitrogen content and X-ray diif~ction data. 3. The presence of tryptophan in the shrimp collagen molecule is disclosed and its significance discussed. 4. Comparisons between the imino acid content of shrimp collagen and that of other collagens are made. INTRODUCTION ALTHOUGH records of amino acid composition of the collagen of a variety of vertebrate and invertebrate species are quite prevalent in the literature, no data have been published on the amino acid composition of the collagen of white shrimp (Penaeus setiferous). In recent years complete amino acid analyses have been obtained on eight different invertebrate collagens from five different invertebrate phyla. However, the amino acid analysis of an Arthropoda collagen is not among the eight reported. X-ray studies of the subcuticular tissues of the lobster and of the peduncle of the barnacle (Lepas) displayed the wide angle pattern of collagen (RudaU, 1955). According to Rudall (1955), Schrnitt, Bear & Clark were able to demonstrate the presence of collagen while conducting a study of crab nerve. Thus, collagen is not in doubt as a connective tissue element in crustaceans. The purposes of this paper are to give the methodology involved in the separation and identification of insoluble shrimp collagen, to report the results obtained in physical and chemical determinations and to report the amino acid composition of insoluble shrimp collagen. MATERIALS AND METHODS Shrimp
Live white shrimp were obtained from a local fishery. They were of such size as to count approximately 60 to the pound. 127
128
HAROLD C. THOMPSON, JR. AND MARY H. THOMPSON
Buffers and chemicals All buffers were prepared using reagent grade chemicals and deionized water.
Isolation of shrimp collagen Most of the methods for the isolation and purification of insoluble collagens are essentially the same. T h e method that was used for the isolation of purified insoluble shrimp collagen was a modification of the procedure of Veis et al. (1960). No other methods were tried since this modified method served adequately. T h e only modification we employed involved homogenizing the shrimp tails with a cold 10% NaCI solution to better facilitate the extraction of the salt soluble impurities. All manipulations were carried out in a cold room maintained at 3°C and in a refrigerated centrifuge also maintained at 3°C. T h e shrimp were obtained live and brought to the laboratory in this condition. They were then headed, peeled and divided into two 140-g portions. T h e duplicate samples were homogenized separately in a Waring Blendor* with 1400 ml of cold 10% NaCI. The two homogenized samples were allowed to stand overnight. T h e suspensions were centrifuged at 8000 rev/min for 20 min, and the supernatants discarded. T h e residues were washed free of chlorides with cold deionized water, and the NaCI extraction was then repeated. Each of the residues was then dehydrated with 300 ml of cold acetone. T h e acetone was decanted, and the residues were extracted four times each with 400 ml of ether. T h e ether was decanted, and the residual ether was removed with cold deionized water. T h e residues were washed four or five times with water to ensure removal of the acetone. T h e residues were then allowed to stand in cold water overnight. T h e suspensions were centrifuged at 8000 rev/min for 20 min, and the supernatants decanted and discarded. T h e residues were each extracted overnight with 500 ml of p H 8"0 phosphate buffer. T h e suspensions were centrifuged at 8000 rev/min for 20 min, and the supernatants were decanted and discarded. The residues were washed free of phosphate buffer with cold deionized water. Each residue was then extracted overnight with 500 ml of p H 3"5 phosphate citrate buffer. The suspensions were centrifuged, and the supernatants were discarded. The residues were washed free o f p H 3"5 buffer with cold deionized water, and were carried through the p H 8'0 and p H 3"5 extractions a second time with a thorough water washing after each extraction. The water suspensions were centrifuged at 8000 rev/min for 20 min, and the water was decanted. T h e residues were then frozen in a bath of dry ice and acetone, and were lyophilized. These samples were stored at 0°F until required for analysis.
Amino acid analysis Each of the duplicate collagen preparations was analyzed for amino acids once. T h e acid hydrolysates were prepared by placing approximately 0"05 g of lyophilized shrimp collagen in 1 ml of 6 N HCI, were evacuated to 30/~ Pig pressure, and were digested at 110°C for 22 hr (Moore & Stein, 1963). T h e hydrolysates were flash evaporated, and the amino acids were redissolved in sodium citrate buffer at p H 2.2 and were stored at 0°F until required for analysis. Factors (obtained by extrapolating data obtained from duplicate samples digested similarly for 22, 48 and 72 hr) were applied in subsequent calculations in order to relate the concentration of the amino acids to the zero time state (Light & Smith, 1963). Amino acids (other than for tryptophan) were analyzed for by the 30-50 ° method using a Beckman Model 120B Amino Acid Analyzer.
Tryptophan determination Tryptophan was determined in accordance with the procedure of Graham et al. (1947).
Hexose determination Hexoses were determined by the method of Seifter et al. (1950) using glucose as a standard. * Use of trade names is merely to facilitate descriptions; no endorsement is implied.
THE COLLAGENOF WHITE 8HRIMP---I
129
Amide nitrogen determination The amide nitrogen value for insoluble shrimp collagen was obtained by hydrolyzing the collagen in N HISO4 for 6 hr, followed by a microKjeldahl distillation and titration.
Total nitrogendetermination Total nitrogen was determined by the microKjeldald method using HgO as a catalyst.
X-ray diffraction X-ray diffraction photographs were taken with a Norelco Debye-Scherrer powder camera, 114"6-mm diameter using Cuka radiation. RESULTS
Yield of collagen Shrimp tails, 140 g (wet basis), yielded 1.8 g of lyophilized insoluble shrimp collagen for each preparation.
General chemical characterization Some general chemical characteristics of the collagen of white shrimp are recorded in Table 1. The total nitrogen content of the preparation was 13.9 per cent, the hexose content was 0.3 residues/1000 total residues, and the amide nitrogen content was 34"2 residues/1000 total residues. TABLE 1----CoNTENTOF SELECTEDCH]gMICALcoMPONENTSOF SrmIMP COLLAO~ (aEsmuBs/1000 TOTALRESIDUES) Total nitrogen
Hexoses
Amide nitrogen
13"9"
0"3
34"2
* Total nitrogen expressed as percentage dry weight.
Amino acid composition The overall amino acid composition of the collagen of white shrimp is listed in Table 2. This collagen exhibits a lysine content of 76-9 residues/1000 total residues, a hydroxylysine content of 8.7 residues/1000 total residues and a glycine content of 149.8 residues/1000 total residues. Shrimp collagen contains tryptophan in the amount of 119-3 residues/1000 total residues. A lower than usual proline content of 66.7 residues/1000 total residues and a lower than usual #-hydroxyproline content of 33"8 residues/1000 total residues are also found. Shrimp collagen exhibits a hydroxylysine : 4-hydroxyproline ratio of 0.26.
X-ray &'ffraction X-ray diffraction patterns of the shrimp collagen fiber showed that this protein is typical of collagen in that it has periodicity. The measurements of the reflection 5
130
HAROLD C. THOMPSON, JR. AND MARY H. THOMPSON
angles showed that the shrimp collagen fiber has a backbone spacing of 4.3 A, a side-chain spacing of 10.0 A, and a repeat distance along the fiber axis of 2.9 A. TABLE 2--AMXNO ACID COMPOSITION OF SHRIMP COLLAGEIq (aesmuss/1000 TOTAL RBsmuRs)* Amino acid 4-Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cystine/2 Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine Tryptophan
Shrimp collagen 33"8
62.2 35"4 43.8 92"8 66.7 149"8 57.7 37.1 3'8 19"8 33"4 51"4 17'5 19.7 8'7 76'9 14.8 55"3 119.3
* The values presented are averages of single amino acid analyses of duplicate preparatiorl$.
DISCUSSION The shrimp obtained for this study were kept alive until they were headed and peeled for use. Iced shrimp from commercial sources have been shown to have connective tissue damage due to bacterial-enzymatic degradation within a few days after being caught by trawling (Love & Thompson, 1966). The use of live shrimp as preparative material reduced the possibility of obtaining degraded component contaminants in the preparation. Shrimp collagen is an anomalous connective tissue protein. It displays the highest lysine content of any reported collagen with a value of 76.9 residues/1000 total residues. Shrimp collagen also exhibits a higher tyrosine, phenylalanine and histidine content than does any other collagen mentioned in the literature, with the single exception of aceUular bovine glomeruli collagen. Glomeruli collagen is reported to contain these amino acids in amounts of 22.8, 32.4 and 17.8 residues/ 1000 total residues, respectively (Lidsky et al., 1967). Shrimp collagen contains these amino acids in amounts of 17.5, 19.7 and 14.8 residues/1000 total residues, respectively. Shrimp collagen exhibits a proline and hydroxyproline content of
131
THE COLLAGEN OF WHITE SHRIMP-"-I
66.7 and 33.8 residues/1000 total residues, respectively. These levels are much lower than are usually found in most other collagens. Shrimp collagen displays a glycine content of 149.8 residues/1000 total residues and an alanine content of 57.7 residues/1000 total residues, whereas most other collagens contain twice that much glycine and s!A_rfine. T h e arginine content of 55.3 residues/1000 total residues for shrimp collagen is approximately the same as that of other collagens. A review of the literature failed to disclose any report of the composition of a collagen whose amino acid pattern contained tryptophan. Shrimp collagen, however, contains 119.3 residues of tryptophan/1000 total residues. Since shrimp collagen does contain a significant amount of tryptophan, it is apparent that this amino acid has taken the place of other amino acid residues. The replaced residues are probably those o f the imino acids, since they are present in about one-half the amounts normally displayed by most collagens. Tryptohan contains a pyrollidinetype moiety that differs from the pyrollidine moiety of proline and hydroxyproline only by unsaturation. This similarity makes it conceivable that a tryptophan residue could take the place of an imino acid in a peptide chain of the shrimp collagen molecule. When the number of residues of tryptophan, proline and hydroxyproline in shrimp collagen are summed, a total of 219.8 residues/1000 total residues is obtained. This total sum falls within the range in total imino acid content of 200-221 residues/1000 total residues reported for many other collagens in the literature (Table 3). TABLE 3 - - I M I N O ACID COMPOSITION OF COLLAGENS FROM DIFFERENT ORIGinS (RESIDUES/1000 TOTAL RESIDUES)
Imino acid
Shrimp tail
Calf skin*
Human skint
E. occidentalis~
B. antiqus~
Hydroxyproline Proline Tryptophan §
33.8 66.7 119"3
85"1 135.5 .
93 128
95 124
99 115
Total
219.8
220.6
219
214
.
. 221
Western horse Ancient bison
.
* Data from Picz et al. (1960). t Data from Bornstein & Piez (1964). ++Data from Ho (1967). § Tryptophan was included under the imino acid heading so that its value could be added to the imino acid total for shrimp tail. Amino acid composition data taken from a recent publication by Lidsky et al. (1967) describing the isolation and amino acid composition of acellular bovine glomeruli collagen provided a striking comparison with the amino acid composition of shrimp collagen. For example, the glycine content of acellular bovine glomeruli collagen and shrimp collagen are of the same order of magnitude, as is demonstrated by a comparison of 162.7 residues/1000 total residues and 149-8 residues/ 1000 total residues, respectively. The proline content of the two collagens is also
132
HAROLDC. THOMPSON,JR. AND i~IARYH. THOMPSON
of the same order of magnitude, with glomeruli collagen exhibiting 59.0 residues of proline/1000 total residues and shrimp collagen exhibiting 66.7 residues of proline/1000 total residues. The amounts of arginine, methionine and isoleucine of shrimp collagen and glomeruli collagen are also similar. There would appear to be no presently known biological basis for the similarity of composition of these two collagens. The similarity is reported merely to show that it does exist. The staining properties of shrimp collagen are somewhat different from those of vertebrate collagen according to the results of previous histological comparisons (Love & Thompson, 1966). When shrimp collagen is stained with Mallory's tripte connective tissue differential stain, a blue to magenta color results. Normally, the color that results is a truer blue. Since this stain depends upon the deposition of tungsten upon the imino residues, the diminished number of residues would result in a poorly defined shade of color. According to yon Hippel & Wong (1963), the ratio of proline to hydroxyproline for most collagens is 1.5 _+0-3. Shrimp collagen is also different with respect to this ratio, for it exhibits a ratio of proline to hydroxyproline of 2-0. With the exception of the presence of tryptophan, decreased amounts of proline and hydroxyproline, and staining properties shown here, shrimp collagen is most typical. REFERENCES BORNSTSINP. & PIEZ K. A. (1964) A biochemical study of human skin collagen and the relation between intra- and intermolecular cross-linking. ~. Clin. Invest. 43, 1813-1823. GRAHAMC. E., SMITHE. P., HIER S. W. & KLEIN D. (1947) An improved method for the determination of tryptophan with p-dirnethylaminobenzaldchyde. ~7. biol. Chem. 168, 711. Ho TONO-YUN (1967) Relationship between imino acid contents of mammalian bone collagen and body temperature as a basis for estimation of body temperature of preldstoric mammals. Comp. Biochem. Physiol. 22, 113-119. LmSKY M. D., SHARPJ. T. & Rtrvnn M. L. (1967) Studies on acellular bovine glomeruli: Isolation, chemical composition, and demonstration of collagen with an unusual hydroxylysine : hydroxyproline ratio. Archs Biochem. Biophys. 121, 491-501. LIOHT A. & SMITH E. L. (1963) Amino acid analysis of peptides and proteins. In The Proteins (Edited by NnORATHH.), Vol. 1, pp. 2-53. Academic Press, New York. L o w T. D. & THOMPSONM. H. (1966) Iced shrimp storage study. Annual Report, Bureau of Commercial Fisheries Technological Laboratory, Pascagoula, Mississippi, fiscal year 1965. U.S. Fish Wildl. Serv. Circ. 251, 15-23. MOORES. & STEINW. H. (1963) Chromatographic determination of amino acids by the use of automatic recording equipment. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLANN. O.), Vol. 6, pp. 819-831. Academic Press, New York. PIEz K. A., Wmss E. & Lmws M. S. (1960) The separation and characterization of the c~- and E-components of calf skin collagen. ~t. biol. Chem. 235, 1987-1991. RUVALLK. M. (1955) The distribution of collagen and chitin. Syrup. Soc. exp. Biol. 9, 49-71. SEI~m~ S., DAYTONS., NovIc B. & M V N ~ R E. (1950) The estimation of glycogen with the anthrone reagent. /Irchs Biochem. Biophys. 25, 191-200. V]~Is A., ANlmEYJ. & COHXNJ. (1960) The depolymerization of collagen fibers..7. Am. Leath. Chem. Ass. 55, 548-563. YON Hn'X~SLP. H. & WONG KwoK-YmQ (1963) Collagen~ gelatin phase transition--I. Further studies of the effects of solvent environment and polypeptide chain composition. Biochemistry 2, 1387-1398.
ISOLATION AND AMINO ACID COMPOSITION OF THE COLLAGEN OF WHITE SHRIMP
(PENAEUS SETIFEROUS)--I H A R O L D C. T H O M P S O N , JR. and MARY H. T H O M P S O N Teclmological Laboratory, Bureau of Commercial Fisheries, Pascegoula, Mis,,itmippi 39567, U.S.A. (Received 22 March 1968)
Atmtract--1. The methodology employed in isolating the insoluble collagen of white shrimp (Penaeus setiferous) is reported. 2. The physical and chemical characteristics of the isolated protein are discussed including amino acid composition, total nitrogen content, hexose content, amide nitrogen content and X-ray diif~ction data. 3. The presence of tryptophan in the shrimp collagen molecule is disclosed and its significance discussed. 4. Comparisons between the imino acid content of shrimp collagen and that of other collagens are made. INTRODUCTION ALTHOUGH records of amino acid composition of the collagen of a variety of vertebrate and invertebrate species are quite prevalent in the literature, no data have been published on the amino acid composition of the collagen of white shrimp (Penaeus setiferous). In recent years complete amino acid analyses have been obtained on eight different invertebrate collagens from five different invertebrate phyla. However, the amino acid analysis of an Arthropoda collagen is not among the eight reported. X-ray studies of the subcuticular tissues of the lobster and of the peduncle of the barnacle (Lepas) displayed the wide angle pattern of collagen (RudaU, 1955). According to Rudall (1955), Schrnitt, Bear & Clark were able to demonstrate the presence of collagen while conducting a study of crab nerve. Thus, collagen is not in doubt as a connective tissue element in crustaceans. The purposes of this paper are to give the methodology involved in the separation and identification of insoluble shrimp collagen, to report the results obtained in physical and chemical determinations and to report the amino acid composition of insoluble shrimp collagen. MATERIALS AND METHODS Shrimp
Live white shrimp were obtained from a local fishery. They were of such size as to count approximately 60 to the pound. 127
128
HAROLD C. THOMPSON, JR. AND MARY H. THOMPSON
Buffers and chemicals All buffers were prepared using reagent grade chemicals and deionized water.
Isolation of shrimp collagen Most of the methods for the isolation and purification of insoluble collagens are essentially the same. T h e method that was used for the isolation of purified insoluble shrimp collagen was a modification of the procedure of Veis et al. (1960). No other methods were tried since this modified method served adequately. T h e only modification we employed involved homogenizing the shrimp tails with a cold 10% NaCI solution to better facilitate the extraction of the salt soluble impurities. All manipulations were carried out in a cold room maintained at 3°C and in a refrigerated centrifuge also maintained at 3°C. T h e shrimp were obtained live and brought to the laboratory in this condition. They were then headed, peeled and divided into two 140-g portions. T h e duplicate samples were homogenized separately in a Waring Blendor* with 1400 ml of cold 10% NaCI. The two homogenized samples were allowed to stand overnight. T h e suspensions were centrifuged at 8000 rev/min for 20 min, and the supernatants discarded. T h e residues were washed free of chlorides with cold deionized water, and the NaCI extraction was then repeated. Each of the residues was then dehydrated with 300 ml of cold acetone. T h e acetone was decanted, and the residues were extracted four times each with 400 ml of ether. T h e ether was decanted, and the residual ether was removed with cold deionized water. T h e residues were washed four or five times with water to ensure removal of the acetone. T h e residues were then allowed to stand in cold water overnight. T h e suspensions were centrifuged at 8000 rev/min for 20 min, and the supernatants decanted and discarded. T h e residues were each extracted overnight with 500 ml of p H 8"0 phosphate buffer. T h e suspensions were centrifuged at 8000 rev/min for 20 min, and the supernatants were decanted and discarded. The residues were washed free of phosphate buffer with cold deionized water. Each residue was then extracted overnight with 500 ml of p H 3"5 phosphate citrate buffer. The suspensions were centrifuged, and the supernatants were discarded. The residues were washed free o f p H 3"5 buffer with cold deionized water, and were carried through the p H 8'0 and p H 3"5 extractions a second time with a thorough water washing after each extraction. The water suspensions were centrifuged at 8000 rev/min for 20 min, and the water was decanted. T h e residues were then frozen in a bath of dry ice and acetone, and were lyophilized. These samples were stored at 0°F until required for analysis.
Amino acid analysis Each of the duplicate collagen preparations was analyzed for amino acids once. T h e acid hydrolysates were prepared by placing approximately 0"05 g of lyophilized shrimp collagen in 1 ml of 6 N HCI, were evacuated to 30/~ Pig pressure, and were digested at 110°C for 22 hr (Moore & Stein, 1963). T h e hydrolysates were flash evaporated, and the amino acids were redissolved in sodium citrate buffer at p H 2.2 and were stored at 0°F until required for analysis. Factors (obtained by extrapolating data obtained from duplicate samples digested similarly for 22, 48 and 72 hr) were applied in subsequent calculations in order to relate the concentration of the amino acids to the zero time state (Light & Smith, 1963). Amino acids (other than for tryptophan) were analyzed for by the 30-50 ° method using a Beckman Model 120B Amino Acid Analyzer.
Tryptophan determination Tryptophan was determined in accordance with the procedure of Graham et al. (1947).
Hexose determination Hexoses were determined by the method of Seifter et al. (1950) using glucose as a standard. * Use of trade names is merely to facilitate descriptions; no endorsement is implied.
THE COLLAGENOF WHITE 8HRIMP---I
129
Amide nitrogen determination The amide nitrogen value for insoluble shrimp collagen was obtained by hydrolyzing the collagen in N HISO4 for 6 hr, followed by a microKjeldahl distillation and titration.
Total nitrogendetermination Total nitrogen was determined by the microKjeldald method using HgO as a catalyst.
X-ray diffraction X-ray diffraction photographs were taken with a Norelco Debye-Scherrer powder camera, 114"6-mm diameter using Cuka radiation. RESULTS
Yield of collagen Shrimp tails, 140 g (wet basis), yielded 1.8 g of lyophilized insoluble shrimp collagen for each preparation.
General chemical characterization Some general chemical characteristics of the collagen of white shrimp are recorded in Table 1. The total nitrogen content of the preparation was 13.9 per cent, the hexose content was 0.3 residues/1000 total residues, and the amide nitrogen content was 34"2 residues/1000 total residues. TABLE 1----CoNTENTOF SELECTEDCH]gMICALcoMPONENTSOF SrmIMP COLLAO~ (aEsmuBs/1000 TOTALRESIDUES) Total nitrogen
Hexoses
Amide nitrogen
13"9"
0"3
34"2
* Total nitrogen expressed as percentage dry weight.
Amino acid composition The overall amino acid composition of the collagen of white shrimp is listed in Table 2. This collagen exhibits a lysine content of 76-9 residues/1000 total residues, a hydroxylysine content of 8.7 residues/1000 total residues and a glycine content of 149.8 residues/1000 total residues. Shrimp collagen contains tryptophan in the amount of 119-3 residues/1000 total residues. A lower than usual proline content of 66.7 residues/1000 total residues and a lower than usual #-hydroxyproline content of 33"8 residues/1000 total residues are also found. Shrimp collagen exhibits a hydroxylysine : 4-hydroxyproline ratio of 0.26.
X-ray &'ffraction X-ray diffraction patterns of the shrimp collagen fiber showed that this protein is typical of collagen in that it has periodicity. The measurements of the reflection 5
130
HAROLD C. THOMPSON, JR. AND MARY H. THOMPSON
angles showed that the shrimp collagen fiber has a backbone spacing of 4.3 A, a side-chain spacing of 10.0 A, and a repeat distance along the fiber axis of 2.9 A. TABLE 2--AMXNO ACID COMPOSITION OF SHRIMP COLLAGEIq (aesmuss/1000 TOTAL RBsmuRs)* Amino acid 4-Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cystine/2 Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine Tryptophan
Shrimp collagen 33"8
62.2 35"4 43.8 92"8 66.7 149"8 57.7 37.1 3'8 19"8 33"4 51"4 17'5 19.7 8'7 76'9 14.8 55"3 119.3
* The values presented are averages of single amino acid analyses of duplicate preparatiorl$.
DISCUSSION The shrimp obtained for this study were kept alive until they were headed and peeled for use. Iced shrimp from commercial sources have been shown to have connective tissue damage due to bacterial-enzymatic degradation within a few days after being caught by trawling (Love & Thompson, 1966). The use of live shrimp as preparative material reduced the possibility of obtaining degraded component contaminants in the preparation. Shrimp collagen is an anomalous connective tissue protein. It displays the highest lysine content of any reported collagen with a value of 76.9 residues/1000 total residues. Shrimp collagen also exhibits a higher tyrosine, phenylalanine and histidine content than does any other collagen mentioned in the literature, with the single exception of aceUular bovine glomeruli collagen. Glomeruli collagen is reported to contain these amino acids in amounts of 22.8, 32.4 and 17.8 residues/ 1000 total residues, respectively (Lidsky et al., 1967). Shrimp collagen contains these amino acids in amounts of 17.5, 19.7 and 14.8 residues/1000 total residues, respectively. Shrimp collagen exhibits a proline and hydroxyproline content of
131
THE COLLAGEN OF WHITE SHRIMP-"-I
66.7 and 33.8 residues/1000 total residues, respectively. These levels are much lower than are usually found in most other collagens. Shrimp collagen displays a glycine content of 149.8 residues/1000 total residues and an alanine content of 57.7 residues/1000 total residues, whereas most other collagens contain twice that much glycine and s!A_rfine. T h e arginine content of 55.3 residues/1000 total residues for shrimp collagen is approximately the same as that of other collagens. A review of the literature failed to disclose any report of the composition of a collagen whose amino acid pattern contained tryptophan. Shrimp collagen, however, contains 119.3 residues of tryptophan/1000 total residues. Since shrimp collagen does contain a significant amount of tryptophan, it is apparent that this amino acid has taken the place of other amino acid residues. The replaced residues are probably those o f the imino acids, since they are present in about one-half the amounts normally displayed by most collagens. Tryptohan contains a pyrollidinetype moiety that differs from the pyrollidine moiety of proline and hydroxyproline only by unsaturation. This similarity makes it conceivable that a tryptophan residue could take the place of an imino acid in a peptide chain of the shrimp collagen molecule. When the number of residues of tryptophan, proline and hydroxyproline in shrimp collagen are summed, a total of 219.8 residues/1000 total residues is obtained. This total sum falls within the range in total imino acid content of 200-221 residues/1000 total residues reported for many other collagens in the literature (Table 3). TABLE 3 - - I M I N O ACID COMPOSITION OF COLLAGENS FROM DIFFERENT ORIGinS (RESIDUES/1000 TOTAL RESIDUES)
Imino acid
Shrimp tail
Calf skin*
Human skint
E. occidentalis~
B. antiqus~
Hydroxyproline Proline Tryptophan §
33.8 66.7 119"3
85"1 135.5 .
93 128
95 124
99 115
Total
219.8
220.6
219
214
.
. 221
Western horse Ancient bison
.
* Data from Picz et al. (1960). t Data from Bornstein & Piez (1964). ++Data from Ho (1967). § Tryptophan was included under the imino acid heading so that its value could be added to the imino acid total for shrimp tail. Amino acid composition data taken from a recent publication by Lidsky et al. (1967) describing the isolation and amino acid composition of acellular bovine glomeruli collagen provided a striking comparison with the amino acid composition of shrimp collagen. For example, the glycine content of acellular bovine glomeruli collagen and shrimp collagen are of the same order of magnitude, as is demonstrated by a comparison of 162.7 residues/1000 total residues and 149-8 residues/ 1000 total residues, respectively. The proline content of the two collagens is also
132
HAROLDC. THOMPSON,JR. AND i~IARYH. THOMPSON
of the same order of magnitude, with glomeruli collagen exhibiting 59.0 residues of proline/1000 total residues and shrimp collagen exhibiting 66.7 residues of proline/1000 total residues. The amounts of arginine, methionine and isoleucine of shrimp collagen and glomeruli collagen are also similar. There would appear to be no presently known biological basis for the similarity of composition of these two collagens. The similarity is reported merely to show that it does exist. The staining properties of shrimp collagen are somewhat different from those of vertebrate collagen according to the results of previous histological comparisons (Love & Thompson, 1966). When shrimp collagen is stained with Mallory's tripte connective tissue differential stain, a blue to magenta color results. Normally, the color that results is a truer blue. Since this stain depends upon the deposition of tungsten upon the imino residues, the diminished number of residues would result in a poorly defined shade of color. According to yon Hippel & Wong (1963), the ratio of proline to hydroxyproline for most collagens is 1.5 _+0-3. Shrimp collagen is also different with respect to this ratio, for it exhibits a ratio of proline to hydroxyproline of 2-0. With the exception of the presence of tryptophan, decreased amounts of proline and hydroxyproline, and staining properties shown here, shrimp collagen is most typical. REFERENCES BORNSTSINP. & PIEZ K. A. (1964) A biochemical study of human skin collagen and the relation between intra- and intermolecular cross-linking. ~. Clin. Invest. 43, 1813-1823. GRAHAMC. E., SMITHE. P., HIER S. W. & KLEIN D. (1947) An improved method for the determination of tryptophan with p-dirnethylaminobenzaldchyde. ~7. biol. Chem. 168, 711. Ho TONO-YUN (1967) Relationship between imino acid contents of mammalian bone collagen and body temperature as a basis for estimation of body temperature of preldstoric mammals. Comp. Biochem. Physiol. 22, 113-119. LmSKY M. D., SHARPJ. T. & Rtrvnn M. L. (1967) Studies on acellular bovine glomeruli: Isolation, chemical composition, and demonstration of collagen with an unusual hydroxylysine : hydroxyproline ratio. Archs Biochem. Biophys. 121, 491-501. LIOHT A. & SMITH E. L. (1963) Amino acid analysis of peptides and proteins. In The Proteins (Edited by NnORATHH.), Vol. 1, pp. 2-53. Academic Press, New York. L o w T. D. & THOMPSONM. H. (1966) Iced shrimp storage study. Annual Report, Bureau of Commercial Fisheries Technological Laboratory, Pascagoula, Mississippi, fiscal year 1965. U.S. Fish Wildl. Serv. Circ. 251, 15-23. MOORES. & STEINW. H. (1963) Chromatographic determination of amino acids by the use of automatic recording equipment. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLANN. O.), Vol. 6, pp. 819-831. Academic Press, New York. PIEz K. A., Wmss E. & Lmws M. S. (1960) The separation and characterization of the c~- and E-components of calf skin collagen. ~t. biol. Chem. 235, 1987-1991. RUVALLK. M. (1955) The distribution of collagen and chitin. Syrup. Soc. exp. Biol. 9, 49-71. SEI~m~ S., DAYTONS., NovIc B. & M V N ~ R E. (1950) The estimation of glycogen with the anthrone reagent. /Irchs Biochem. Biophys. 25, 191-200. V]~Is A., ANlmEYJ. & COHXNJ. (1960) The depolymerization of collagen fibers..7. Am. Leath. Chem. Ass. 55, 548-563. YON Hn'X~SLP. H. & WONG KwoK-YmQ (1963) Collagen~ gelatin phase transition--I. Further studies of the effects of solvent environment and polypeptide chain composition. Biochemistry 2, 1387-1398.