Chemical Papers 66 (7) 636–641 (2012)DOI: 10.2478/s11696-012-0172-0

ORIGINAL PAPER

Analysis and improvement of stability of pepsin-solubilized collagenfrom skin of carp (�������� ����)

aRui Duan*, aJun-Jie Zhang, bKunihiko Konno, aMei-Hua Wu, aJing Li, aYe Chen

aSchool of Marine Science and Technology, Huaihai Institute of Technology, 59 Cangwu Road Lianyungang, 222005, China

bFaculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato Hakodate, Hokkaido 041-8611, Japan

Received 12 September 2011; Revised 31 January 2012; Accepted 1 February 2012

Pepsin is widely used for the extraction of pepsin-solubilized collagens (PSC) from many resources.PSC-A and PSC-P were prepared from carp skin using 0.1 mol L−1 acetic acid and 0.02 mol L−1

Na2HPO4 (pH 7.2) as the dialysis solution, respectively. SDS-PAGE patterns showed PSC-A andPSC-P as type I collagens, as well as acid soluble collagen (ASC). When incubated at 40◦C, nodegradation was observed for ASC, but PSC-A and PSC-P were degraded into short peptides,showing lower stability than ASC. The results indicate that pepsin remaining in the PSCs resultedin their degradation, which was confirmed by the inhibition using pepstatin. This research revealedthe behavior of the remaining pepsin in pepsin-solubilized collagens and an approach to the PSCstability improvement was proposed. Chromatography profiles showed that new PSC prepared bythe improved method had almost the same stability as ASC.c© 2012 Institute of Chemistry, Slovak Academy of Sciences

Keywords: carp, collagen, pepsin, degradation, dialysis, Cyprinus carpio

Introduction

Collagen has a wide range of applications inbiomedical materials, pharmaceutical, food, cosmetic,leather, and film industries (Bailey & Light, 1989;Cavallaro et al., 1994; Hood, 1987). Pepsin treatmentcan be used for extraction of insoluble collagen fromtissues (Neurath et al., 1975). Pepsin digestion is usu-ally limited to non-triple-helical terminal extensions(Bornstein et al., 1966). Compared to ASC, pepsin-solubilized collagen (PSC) has low antigenic proper-ties because of the loss of N- and C-terminus domainsin the molecule of PSC (Ikoma et al., 2003).Researchers usually prepare PSCs by employing

0.1 mol L−1 acetic acid or 0.02 mol L−1 Na2HPO4(pH 7.2) as the dialysis solution for collagen purifica-tion; also properties of PSCs were reported (Ogawaet al., 2004; Mizuta et al., 2002a, 2002b; Nagai et al.,2000, 2002). However, no information regarding theinfluence of the remaining pepsin and dialysis solu-tion on the stability of pepsin-solubilized collagen has

been reported, especially for collagens from carp skin.In this research, pepsin-solubilzed collagens (PSC-

A and PSC-P) were prepared using 0.1 mol L−1 aceticacid and 0.02 mol L−1 Na2HPO4 as the dialysis solu-tion, respectively. The stability of PSCs from carp skinwas studied and the method of stability improvementwas investigated.

Experimental

Live cultured carps (Cyprinus carpio) were ob-tained from a free market in Lianyungang, Jiangsuprovince, in July 2010. The live fish (ten individu-als; body mass of (803 ± 30) g; length of the sample(47 ± 3) cm) were transported to the laboratory. Theskins were removed manually and washed with chilledtap water. The samples were then placed in polyethy-lene bags and stored at –25◦C until their use.All reagents were of analytical grade and were ob-

tained fromWako Pure Chemical Industries, Ltd. (Os-aka, Japan) or Sigma–Aldrich (St. Louis, MO, USA).

*Corresponding author, e-mail: duanrui526@yahoo.com.cn

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The water used was distilled and deionized using aMillipore “Super Q” system.The collagens were prepared by the method of

Nagai and Suzuki (2000) with a slight modification(Duan et al., 2009). All preparation procedures wereperformed at 4◦C.The amount of 1 g of skins was mixed with 8 mL

of 0.1 mol L−1 NaOH to remove non-collagenous pro-teins. The mixture was stirred for 6 h and the alkalisolution was changed every 3 h. Then, the sampleswere washed with cold distilled water until neutral pHof washing water was obtained.Deproteinized skins were soaked in butyl alco-

hol/water (ϕr = 10 vol. %) with a solid/solvent ratioof 1 g : 10 mL overnight to extract fat and then, thesamples were washed with cold distilled water repeat-edly. The treated skins were cut into small pieces byscissors and extracted with 0.5 mol L−1 acetic acid forthree days under stirring. The extract was centrifugedat 20000g for 1 h. Supernatants were salted out byadding NaCl to the final concentration of 2.5 mol L−1

in the presence of 0.05 mol L−1 Tris (2-amino-2-hydroxymethyl-propane-1,3-diol). The resultant pre-cipitate was collected by centrifugation at 20000g for30 min. The pellet was dissolved in 0.5 mol L−1 aceticacid, dialyzed against 0.1 mol L−1 acetic acid and dis-tilled water and then it was lyophilized.After the skins were extracted with 0.5 mol L−1

acetic acid, their insoluble components were solubi-lized with 10 volumes of 0.5 mol L−1 acetic acid con-taining 1 g L−1 of pepsin (1/10000, Sigma–Aldrich,St. Louis, MO, USA) for three days. The solution wascentrifuged at 20000g for 30 min at 4◦C. Supernatantswere salted out from the extract by adding NaCl togive the final concentration of 2.5 mol L−1 in the pres-ence of 0.05 mol L−1 Tris. The resulting precipitatewas collected by centrifugation at 20000g for 30 minand then dissolved in 5 volumes of 0.5 mol L−1 aceticacid. The procedures for salting-out and solubilizationwere repeated three times.The resulting solution was separated into two equal

volumes. One was dialyzed against 0.1 mol L−1 aceticacid and distilled water for three days, respectively.The dialysis solution was changed five times every day.Then, the sample was lyophilized (pepsin-solubilizedcollagen; PSC-A). The other was dialyzed against0.02 mol L−1 Na2HPO4 (pH 7.2) and distilled wa-ter for three days, respectively. The dialysis solutionwas changed five times every day. Then, the samplewas lyophilized (pepsin-solubilized collagen; PSC-P).SDS-PAGE (sodium lauryl sulfate–polycrylamide

gel electrophoresis) was performed by the method ofLaemmli (1970). Collagen samples were dissolved in0.1 mol L−1 acetic acid, mixed with the sample buffer(0.5 mol L−1 Tris–HCl, pH 6.8, containing 5 g L−1 ofSDS, and 20 g L−1 of glycerol) at the ratio of 1 : 2in the presence of β-ME (2-sulfanylethan-1-ol, ϕr =10 vol. %). Electrophoresis was performed on 7.5 %

(g mL−1) gels. High molecular mass markers (Sigma–Aldrich, St. Louis, MO, USA) were used to estimatethe molecular mass of proteins; for this purpose, 10 µgof protein was loaded in each well.To study the thermo stability of ASC and PSC-A,

lyophilized collagens were dissolved in 0.1 mol L−1

acetic acid in the concentration of 2 mg mL−1 at4◦C. The collagens were kept at different temperatures(from 15◦C to 40◦C) for 5 h. Then, half the volumeof the sample buffer (0.5 mol L−1 Tris–HCl, pH 6.8,containing 5 g L−1 SDS, 20 g L−1 glycerol, and 10 %β-ME) was added to the samples and heated imme-diately in boiling water for 2 min. The samples wereresolved on 7.5 % polyacrylamide gels containing 0.1 %(g mL−1) of SDS (SDS-PAGE) (Laemmli, 1970). Pro-tein in the amount of 10 µg was loaded in each well.The patterns of ASC and PSC-A were compared.To compare the stability of collagens, ASC, PSC-

A, and PSC-P, were dissolved in 0.1 mol L−1 aceticacid in the concentration of 2 mg mL−1 at 4◦C, respec-tively, and incubated at 40◦C. At intervals, aliquotsof the collagens were withdrawn and terminated byadding half the volume of the sample buffer (0.5mol L−1 Tris–HCl, pH 6.8, containing 5 g L−1 of SDS,20 g L−1 of glycerol, and 10 % of β-ME). Then, thesamples were heated immediately in boiling water for2 min. The digests were also resolved on 7.5 % SDS-PAGE.Subunit components of carp skin collagens were

separated by a SP-Toyopearl 650M (Tosoh Co. Tokyo,Japan) column chromatograph. According to themethods of Mizuta et al. (2002) and Kimura & Ohno(1987), a 10 mg ASC sample was dissolved in 5 mL ofa 20 mmol L−1 sodium acetate buffer, pH 4.8, at 4◦Covernight, and denatured at 45◦C for 30 min. Aftercentrifugation at 20◦C for 30 min, the denatured colla-gen was fractionated on a column of the SP-Toyopearl650 mol L−1 (1.5 cm× 10 cm) chromatograph. Elutionwas achieved using the starting buffer with the lineargradient of 100–250 mmol L−1 of NaCl over the totalvolume of 300 mL at the flow rate of 90 mL h−1. Ap-propriate fractions were pooled. Fractions indicatedby numbers were examined by SDS-PAGE.

Results and discussion

Electrophoresis

The PSC-A and PSC-P, as well as the ASC samplesfrom carp skin were analyzed by SDS-PAGE using a7.5 % gel (Fig. 1).The SDS-PAGE pattern showed that ASC and

PSCs were almost the same. They were all composedof at least two different α chains, α1 and α2. The den-sity of α1 is higher than that of α2. It was suggestedthat they were type I collagens. The two α chains ofASC and PSCs were similar in the electrophoretic mo-bility suggesting that the α chains of ASC and PSCs

638 R. Duan et al./Chemical Papers 66 (7) 636–641 (2012)

Fig. 1. SDS-PAGE of ASC and PSCs from carp skin on a 7.5 %gel. Carp skin ASC (1); carp skin PSC-A (2); carp skinPSC-P (3); high molecular mass marker (4).

have similar molecular mass. The cleavage sites ofPSCs by pepsin were located very close to the ter-minal ends of the tropocollagen (Ogawa et al., 2003).Molecular mass of α2 was 116 kDa. A great number ofβ chains can be observed in the pattern of the colla-gens. There is no discrepancy in the amount of β andγ components of ASC and PSCs.

Thermal stability of ASC and PSCs

To reveal the differences in thermal stability ofASC and PSC, the SDS-PAGE patterns of ASC andPSC-A incubated at different temperatures for 5 hwere compared. Fig. 2 shows that PSC-A and ASCmaintained integrate structures when the incubationtemperature was below 28◦C. However, the degrada-tion of PSC-A was obvious at the temperature of 28◦C.Acid soluble collagen showed no fragmentation of thesubunit components at 30◦C and 40◦C, while PSC-Awas degraded into many short peptides at tempera-tures above 28◦C.Therefore, there must be some factors causing the

degradation of PSC-A. It was inferred that pepsinused for the extraction of PSC-A remained in thelyophilized samples, although salting-out by NaCl wascarried out three times for PSC-A purification. Sincethe molecular mass of pepsin is about 34 kDa, it ispossible that it was salted out with collagens in thisprocedure. The amount of the remaining pepsin canbe very small but it still has digestion ability undersuitable conditions (around 37◦C). Extensive studieson vertebrate collagens have demonstrated that thetriple-helical conformation of native collagen is resis-tant to the degradation by most proteinases exceptspecific collagenases (Gross & Lapiere, 1962). The re-sults show that when incubated at above 28◦C, PSC-Adenatures gradually and it can be digested by the re-maining pepsin.To confirm this assumption, the inhibitor of pepsin,

Fig. 2. SDS-PAGE patterns of carp skin ASC and PSC-A in-cubated at various temperatures. ASC (A) and PSC-A (B) were dissolved in 0.1 mol L−1 acetic acid, re-spectively, and incubated for 5 h at different tempera-tures from 15◦C to 40◦C. At intervals, aliquots of col-lagen were withdrawn and then resolved on 7.5 % SDS-PAGE.

Fig. 3. SDS-PAGE patterns of PSC-A and PSC-A with pep-statin incubated at 40◦C for 5 h. Carp skin PSC-A (1);carp skin PSC-A with pepstatin (2).

pepstatin, was employed. The amount of 2 mg of PSC-A was dissolved in 1 mL of 0.1 mol L−1 acetic acid at4◦C, then, 10 µL of 10 mmol L−1 pepstatin (Sigma–Aldrich, St. Louis, MO, USA) were added. The sam-ple was incubated at 40◦C for 5 h. As shown in theSDS-PAGE pattern, no PSC-A degradation was ob-served when pepstatin was added (Fig. 3). The resultsdemonstrated that it was pepsin remaining in PSC-Awhich caused the destruction of the collagen structure.The samples of PSC-A and PSC-P, when incu-

bated at 40◦C, were all degraded (Fig. 4B). However,degradation of PSC-A was more severe than that ofPSC-P. In the incubation time of 1 h, α and β compo-nents of PSC-A disappeared almost completely, whileonly a small amount of fragments was detected in thepattern of PSC-P. At the same time, acid soluble col-

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Fig. 4. SDS-PAGE patterns of ASC (A), PSC-P (B), and PSC-A (C) incubated at 40◦C. Carp skin ASC, 0 h (1); ASC, 0.5 h (2);ASC, 1 h (3); ASC, 2 h (4); ASC, 3 h (5); ASC, 5 h (6); carp skin PSC-P, 0 h (7); PSC-P, 0.5 h (8); PSC-P, 1 h (9); PSC-P,2 h (10); PSC-P, 3 h (11); PSC-P, 5 h (12); carp skin PSC-A, 0 h (13); PSC-A, 0.5 h (14); PSC-A, 1 h (15); PSC-A, 2 h(16); PSC-A, 3 h (17); PSC-A, 5 h (18).

lagen (ASC) was not degraded, showing no fragmen-tation of α and β components (Fig. 4A).Since pepsin is a kind of acid proteinase with the

optimum pH value at around 2, the acid solution wasnot harmful to the remaining pepsin when 0.1 mol L−1

acetic acid was employed as the dialysis solution.PAC-A was degraded into fragments under suitableconditions (Fig. 4C). However, when 0.02 mol L−1

Na2HPO4 (pH 7.2) was used, pepsin was denaturedto some extent in the mild alkali solution resulting inmuch weaker digestion ability under the same exper-imental conditions as shown in Fig. 4B. Pepsin wasnot employed for the extraction of ASC; therefore, nopepsin remained in the sample (Fig. 4A).Some researchers used 0.1 mol L−1 acetic acid and

others employed 0.02 mol L−1 Na2HPO4 (pH 7.2) asthe dialysis solutions for the pepsin-solubilized colla-gen preparation (Ogawa et al., 2004; Mizuta et al.,2002b; Nagai et al., 2002, 2000). However, there is noreport on the degradation caused by pepsin remainingin the PSC samples.

Chromatography

To determine the subunit composition of colla-gens, the denatured collagens were resolved by SP-Toyopearl 650M column chromatography. Collagendissolved in a 60 mmol L−1 sodium acetate buffer washeated at 45◦C for 30 min. Then, the chromatogra-phy was carried out at 40◦C. Various chromatographicfractions were examined by SDS-PAGE and the frac-tions were indicated by numbers.

Fig. 5. SP-Toyopearl chromatography of PSC-P from carpskin, λ = 220 nm (A). Fractions indicated by numberswere examined by 7.5 % SDS-PAGE (B).

Fig. 5 shows the chromatography and SDS-PAGEpatterns of PSC-P. Although the component of thefirst peak had no degradation, the second and thirdpeaks were composed of many degraded fragments.This result is in accordance with that in Fig. 4. Sincepepsin remaining in the PSC-P sample was not dena-tured completely and the chromatography was carriedout at 40◦C, it still had the digestion ability to degradecollagen into short peptides under the experimentalconditions employed.

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Fig. 6. SP-Toyopearl chromatography of new PSC-P from carpskin (A). Fractions indicated by numbers were exam-ined by 7.5 % SDS-PAGE (B). Fraction A: α1 chain;fraction B: α3 chain and its dimers; fraction C: α2 chainand its dimers.

To improve the stability of PSC, some mea-sures were taken to achieve complete denaturation ofpepsin. During the preparation, pH value of the dial-ysis solution was increased to 9–11 providing a newsample of PSC-P (new PSC-P). After the treatment,the result of chromatography of new PSC-P was dif-ferent from that of PSC-P showing no degradation ofsubunit components (Fig. 6). Fig. 6 presents the chro-matography and SDS-PAGE profiles of new PSC-P.As can be seen in the SDS-PAGE pattern, the firstpeak contained only the α1 chain component (frac-tion A), and the second peak was a mixture of α3and β chains. The third peak (fraction C) containedthe α2 component whose molecular mass was lowerthan those of α1 and α3 according to their mobilityon SDS-PAGE. The results are almost identical withthe chromatography and SDS-PAGE profiles of ASC(Fig. not shown here).Therefore, the results indicate that the dialysis so-

lution affected the stability of pepsin-solubilized col-lagens. For PSC-A, degradation can occur at 28◦C.For the PSC-P sample, the remaining pepsin stillshowed digestion ability at 40◦C, resulting in the sam-ple degradation into short peptides. It was suggestedthat although PSCs were purified by salting-out withNaCl three times, a small amount of pepsin still re-mained in the collagen. Dialyzing against 0.1 mol L−1

acetic acid was not sufficient to denature pepsin whichcan degrade the collagens under suitable conditions

(collagen was dissolved in acetic acid solution and keptat above 28◦C). In addition, dialyzing against 0.02 molL−1 Na2HPO4 (pH 7.2) was not sufficient to denaturethe remaining pepsin completely, which can also causethe degradation of PSC (denatured) at 40◦C. Increas-ing the pH value of the dialysis solution resulted in animprovement of the stability of PSC-P. The stabilityof new PSC-P was similar to that of ASC.

Conclusions

Acid soluble and pepsin-solubilized collagens havewide applications in cosmetics, biomedical, and phar-maceutical industries. Pepsin is used for the extractionof collagens from many resources. As the dialysis so-lution for PSC purification, 0.1 mol L−1 acetic acidor 0.02 mol L−1 Na2HPO4 (pH 7.2) are usually em-ployed. However, there is no report on the effects ofthe remaining pepsin and dialysis solution on the sta-bility of PSC.In this study, PSC-A and PSC-P were prepared

from carp skin using 0.1 mol L−1 acetic acid and 0.02mol L−1 Na2HPO4 (pH 7.2) as the dialysis solution,respectively. The results show that pepsin remains inthe PSCs even after their three times repeated pu-rification by salting-out. Furthermore, the remainingpepsin can not be denatured by dialyzing against thetwo solutions. When incubated at 40◦C, no degrada-tion was observed for ASC, but PSC-A and PSC-Pwere degraded into short peptides, showing lower sta-bility than ASC. Therefore, great attention shouldbe paid to the dialysis procedure. It was confirmedthat the stability of PSC can be improved by dia-lyzing against a solution with higher pH value (pH9–11). Chromatography profiles showed that the newPSC prepared by the improved method had almostthe same stability as ASC. The study is of great sig-nificance for the production and utilization of pepsin-soluble high quality collagens.

Acknowledgements. This work was supported by grantsfrom the Japan Society for the Promotion of Science (JSPS),Jiangsu Key Laboratory of Marine Science and Technol-ogy (JSKLMST) through the Research Foundation Project2010HS010, Jiangsu Marine Resource Development ResearchInstitute through Project No. JSIMR09D04. We would like toexpress our gratitude to JSPS, JSKLMST, and JSIMRDR.

References

Bailey, A. J., & Light, N. D. (1989). Connective tissue in meatand meat products (pp. 238–242). London, UK: Elsevier Ap-plied Science.

Bornstein, P., Kang A. H., & Piez, I. A. (1966). The nature andlocation of intramolecular cross-links in collagen. Proceedingsof the National Academy of Sciences, 55, 417–424.

Cavallaro, J. F., Kemp, P. D., & Kraus, K. H. (1994). Collagenfabrics as biomaterials. Biotechnology and Bioengineering,43, 781–791. DOI: 10.1002/bit.260430813.

Duan, R., Zhang, J., Du, X., Yao, X., & Konno, K. (2009).Properties of collagen from skin, scale and bone of carp

R. Duan et al./Chemical Papers 66 (7) 636–641 (2012) 641

(Cyprinus carpio). Food Chemistry, 112, 702–706. DOI:10.1016/j.foodchem.2008.06.020.

Gross, J., & Lapiere, C. M. (1962). Collagenolytic activity inamphibian tissues: a tissue culture assay. Proceedings of theNational Academy of Sciences, 48, 1014–1022.

Hood, L. L. (1987). Collagen in sausage casings. In A. M. Pear-son, T. R. Dutson, & A. J. Bailey (Eds.), Advances in meatresearch (Vol. 4, pp. 109–129). New York, NY, USA: Nos-trand Reinhold.

Ikoma, T., Kobayashi, H., Tanaka, J., Walsh, D., & Mann, S.(2003). Physical properties of type I collagen extracted fromfish scales of Pagrus major and Oreochromis niloticas. Inter-national Journal of Biological Macromolecules, 32, 199–204.DOI: 10.1016/s0141-8130(03)00054-0.

Kimura, S., Ohno Y., Miyauchi Y., & Uchida, N. (1987). Fishskin type I collagen: wide distribution of an α3 subunit inteleost. Comparative Biochemistry and Physiology Part B:Comparative Biochemistry, 88, 27–34. DOI: 10.1016/0305-0491(87)90074-5.

Laemmli, U. K. (1970). Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature, 277,680–685. DOI: 10.1038/227680a0.

Mizuta, S., Hwang, J., & Yoshinaka, R. (2002a). Molecu-lar species of collagen from wing muscle of skate (Rajakenojei). Food Chemistry, 76, 53–58. DOI: 10.1016/s0308-8146(01)00249-7.

Mizuta, S., Miyagi, T., Nishimiya, T., & Yoshinaka, R. (2002b).Partial characterization of collagen in mantle and adductorof pearl oyster (Pinctada fucata). Food Chemistry, 79, 319–325. DOI: 10.1016/s0308-8146(02)00148-6.

Nagai, T., Araki, Y., & Suzuki, N. (2002). Collagen of the skinof ocellate puffer fish (Takifugu rubripes). Food Chemistry,78, 173–177. DOI: 10.1016/s0308-8146(01)00396-x.

Nagai, T., & Suzuki, N. (2000). Isolation of collagen from fishwaste material — skin, bone and fins. Food Chemistry, 68,277–281. DOI: 10.1016/s0308-8146(99)00188-0.

Nagai, T., Worawattanamateekul, W., Suzuki, N., Nakamura,T., Ito, T., Fujiki, K., Nakao, M., & Yano, T. (2000). Isolationand characterization of collagen from rhizostomous jellyfish(Rhopilema asamushi). Food Chemistry, 70, 205–208. DOI:10.1016/s0308-8146(00)00081-9.

Neurath, H., Hill, R. L., & Boeder, C. L. (1975). The proteins.New York, NY, USA: Academic Press.

Ogawa, M., Moody, M. W., Portier, R. J., Bell, J., Schexnay-der, M. A., & Losso, J. N. (2003). Biochemical propertiesof black drum and sheepshead seabream skin collagen. Jour-nal of Agricultural and Food Chemistry, 51, 8088–8092. DOI:10.1021/jf034350r.

Ogawa, M., Portier, R. J., Moody, M. W., Bell, J., Schexnay-der, M. A., & Losso, J. N. (2004). Biochemical properties ofbone and scale collagens isolated from the subtropical fishblack drum (Pogonia cromis) and sheepshead seabream (Ar-chosargus probatocephalus). Food Chemistry, 88, 495–501.DOI: 10.1016/j. foodchem.2004.02.006.