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Purification and characterization of type II collagen
from chick sternal cartilage
Hui Cao, Shi-Ying Xu *
School of Food Science and Technology, Southern Yangtze University, P.O. Box 98, No. 1800, Lihu Road, Wuxi, 214122 Jiangsu, China Received 9 March 2007; received in revised form 10 September 2007; accepted 11 September 2007
Abstract
Type II collagen was purified from sternal cartilage of the chick using a combination of pepsin digestion, NaCl precipitation and DEAE-sepharose CL 6B ion exchange chromatography. Pepsin-solubilized type II collagen of higher stability can be obtained with the extraction time of 32 h, 0.5% pepsin concentration at 20 °C. The purified preparation showed a single peak on RP-HPLC and a single band (a-chain) and its dimers (b-chains) on SDS–PAGE with a subunit Mr of 110 kDa. The amino acid composition of the type II col- lagen derived from chick cartilage was closer to that of reference Sigma–Aldrich type II collagen which contains more imino acid. Anal- ysis by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR) revealed that type II collagen from chick sternal cartilage retains more intermolecular crosslinks during the purification process. Collagen purified from chick sternal car- tilage was typical type II collagen and may find applications in functional foods. Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Type II collagen; Purification; Sternal cartilage; Characterization; Secondary structure
- Introduction
Type II collagen is the main structural component of cartilage and, together with other tissue-specific collagens and proteoglycans, provides the tissue with its shock- absorbing properties and its resiliency to stress (Gelse, Poschl, & Aigner, 2003). Type II collagen with specific molecular structure is used in various food applications (clarification agent, emulsifier, or whipping agent). Its usage extends even further to other industrial (shampoo and lipstick) and pharmaceutical applications (tissue engi- neering material, microencapsulation, or tablet coating). Today, there is an increasing demand for type II collagen as research suggests that type II collagen can suppress Rheumatoid arthritis (RA) and promote healthy joints as superior dietary supplement products (David, Alexander, & Andrew, 1977; David & Roselyn, 1993; Takashi, Akio, & Satoshi, 1998).
Type II collagen, traditionally, has been extracted from bovine or porcine articular cartilage. Chick sternal carti- lage containing a high amount of collagen, one of the by- products of chick manufacturing industry is recognized as a potential source of type II collagen. However, most chick sternal cartilage is conventionally used to produce animal feed or is directly discharged into estuaries resulting in environmental pollution. Thus, new strategies must be explored to find a way of upgrading the processing of waste to value added products such as type II collagen. Type II collagen has been extracted from articular carti- lage. The result indicates that the functional properties of type II collagen are highly influenced by its molecular structure (Miller, 1971; Rigo, Hartmann, & Bairati, 2002). In general, the telopeptide of type II collagen is thought to be responsible for causing an immunogenic response when introduced into xenogenic hosts (Takaoka, Koezuka, & Nakahara, 1991). To eliminate this problem, pepsin has been applied to solubilize collagen and remove telopeptides. Ramesh and Sehgal (1991) described the pro- cedure that involved suspending tissue (200 g wet weigh) in
0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2007.09.
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Food Chemistry 108 (2008) 439–
Food
Chemistry
1.5 L of 0.5 M acetic acid mixed with 100 mg pepsin and incubation at 1 °C for 48 h with stirring. Vasantha, sehgal, and Rao (1988) used 0.001 M hydrochloric acid for prepa- ration of telopeptide-poor collagen by treatment with pep- sin (approximate ratio of enzyme to collagen was 1:400) at 20 °C with intermittent stirring for 5 days. The main diffi- culty, however, with all these techniques, which involve various different digestion conditions of time, temperature and pepsin concentration, is that they can not ensure the quality of the pepsin-solubilized type II collagen isolated from cartilage. Circular dichroism (CD) is particularly use- ful for analyzing collagen and associated degradation prod- ucts, who are able to assign the secondary structure of type II collagen (Ikoma, Kobayashi, Tanaka, Walsh, & Mann, 2003; Usha & Ramasami, 2005). This paper describes the effect of temperature, time and pepsin concentration on the yield and secondary structure of type II collagen, with the aim of producing extracted protein with minimal changes to its functional properties. Further, some biochemical characterizations of type II col- lagen from chick sternal cartilage are also assessed.
- Materials and methods
2.1. Materials
Chick sternal cartilage was provided by Nanjing YuRun Co., Ltd. (Nanjing, China), and stored in refrigerator at 20 °C until use. Resins of DEAE-sepharose CL 6B were purchased from Pharmacia Biotech (Uppsala, Sweden). Standard protein (e.g., myosin heavy chain 200 kDa, Camodulin-binding protein, 130 kDa, Rabbit Phosphory- lase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; rabbit actin, 43 kDa) for sodium dodecyl sulphate–polyacryl- amide gel electrophoresis (SDS–PAGE) were obtained from Shanghai Huamei Biotech (Shanghai, China). Stan- dard type II collagen and pepsin (EC 3.4.23.1) were pur- chased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were in reagent grade or higher.
2.2. Pretreatment of chick sternal cartilage
The sternal cartilage of chicks were cleaned to remove adhering tissue and washed thoroughly with water. The cartilage was cut into small pieces and defatted with chlo- roform–methanol (2:1, v/v). After the pieces of tissue were cleaned with deionized water, then the chloroform–metha- nol-free pieces were stored at 20 °C until use.
2.3. Preparation of pepsin-solubilized type II collagen
2.3.1. Digestion test Sternal cartilage was homogenized at 10,000 rpm for 10 min using 1000 ml 0.2 M NaCl in 0.05 M Tris–HCl (pH 7.5). The mixture was then extracted using 1.0 M NaCl in 0.05 M Tris–HCl (pH 7.5) at 4 °C for 24 h. After the extracts were aggregated by centrifugation at 8000g at
4 °C, the digestion was tested by mixing precipitation with pepsin to assess the effect of temperature (4 °C, 20 °C and 37 °C ), times (16 h, 32 h and 48 h) and the ratio of enzyme to precipitation (1:100, 1:200 and 1:400) on the yield and secondary structure of type II collagen. The resulting vis- cous solution was centrifuged at 10,000g for 30 min to remove insoluble substances. NaCl was added to a final concentration of 0.9 M, and the collagen was allowed to precipitate for 16 h. The precipitated collagen was dis- solved in 0.5 M acetic acid (pH 2.5) and aggregated by dial- ysis against 0.02 M phosphate buffer (pH 7.4), then lyophilized. The lyophilized collagen was stored in desicca- tor placed in a refrigerator (4 °C), until used.
2.3.2. The yield of pepsin-solubilized type II collagen The yield of pepsin-solubilized type II collagen with dif- ferent digestion conditions was monitored by the content of Hydroxyproline. The percentage (%) of hydroxyproline in the collagen was determined using the method of Reddy and Enwemeka (1996).
2.3.3. The secondary structure of pepsin-solubilized type II collagen Circular dichroism (CD) spectra were applied to assess the secondary structure of pepsin-solubilized type II colla- gen from the different digestion conditions. The type II col- lagen was diluted using 0.05 M acetic acid and then the solution placed into a quartz cell with a path length of 1 mm. CD spectra measurements were performed and the wavelengths 250–190 nm with a scan speed of 100 nm/ min at an interval of 1.0 nm. A reference spectrum contain- ing acetic acid was also recorded. The CD spectra of the samples were obtained after subtracting the reference spec- trum. The data were accumulated three times.
2.4. Purification of type II collagen
2.4.1. DEAE-sepharose CL 6B ion exchange chromatography The lyophilized pepsin-solubilized type II collagen was dissolved in 0.05 M acetic acid and dialyzed overnight at 4 °C against 100 volumes of 0.2 M NaCl (0.05 M Tris– HCl, pH 7.5). During dialysis, the solution within the dial- ysis tubing remained clear. Following dialysis, 3 ml of extract were loaded onto the 1 20 cm column of DEAE-sepharose CL 6B equilibrated with 0.2 M NaCl (0.05 M Tris–HCl, pH 7.5). The flow rate of column was maintained 0.6 ml/min. The column effluent was monitored and recorded at 280 nm. After application of the sample to the column, elution with 0.2 M NaCl (0.05 M Tris–HCl, pH 7.5) was continued until no further ultraviolet-absorb- ing material was detected in the effluent. At this time, the eluting solvent was changed to 1.0 M NaCl (0.05 M Tris– HCl, pH 7.5) and elution with the latter buffer was contin- ued until an additional peak was eluted from the column. The column was reequilibrated with the starting buffer and was ready for reuse.
CD-spectra of protein solutions provide information about secondary structure content of proteins (Kelly & Price, 1997; Usha, Maheshwan, Dhathathreyan, & Ramas- ami, 2006). Fig. 1 shows the CD spectra of the pepsin-sol- ubilized type II collagen under different extraction conditions. All collagen samples showed a rotatory maxi- mum at 221 nm (positive band), minimum at 198 nm (neg- ative band) and a consistent cross over point (zero rotation) at about 212 nm, which was characteristic of tri- ple helical conformation of the protein (Usha & Ramas- ami, 2004). The ellipticity of type II collagen deduced from CD spectra are given in Table 1. It is evident that in the presence of increasing time and temperature, there was a decrease in ellipticity at 221 nm and an increase at 198 nm, while the increase of pepsin concentration did not alter the ellipticity significantly. This can be interpreted as being due to decomposition of the collagen triple helical structure with increase in digestion temperature and time. However, it has been reported that on complete denatur-
ation, the positive peak at 221 nm disappears completely and the negative band is found red shifted (Kwak, Jeffer- son, Bhumralkar, & Goodman, 1999). In this investigation, there was neither significant change in the red shift of the negative band nor any disappearance of the positive band at 221 nm. The results indicated that partially denatured collagen gave CD spectra with increasing digestion times and temperature. The secondary structure of the pepsin-solubilized type II collagen with different extraction condition is given in Table 1. The digestion temperature and time have a signif- icant effect on the secondary structure of type II collagen. Extraction at 37 °C resulted in 60.9% random coil com- pared to 53.5% at 4 °C and extraction for 48 h resulted in 60.3% random coil compared to 54.4% for 16 h. Although an increase in digestion time and temperature give higher collagen yields, the longer times and higher temperatures also result in a higher change of collagen secondary struc- ture. This is not desirable since it causes a higher destruc- tion of secondary structure of collagen resulting in altered functional properties. It seemed, therefore, that the pepsin-solubilized type II collagen of higher stability can be obtained with the extraction times for 32 h, pepsin concentration for 0.5% and temperature of 20 °C.
3.2. Purification determination of type II collagen
The purification procedures of type II collagen consisted of chromatography techniques and NaCl precipitation as outlined above in Section 2.4. DEAE-sepharose CL 6B chromatography of neutral-salt extracts of cartilage results in the fractionation of the total extract into two major components. The effluent from both peaks were dialyzed against water and lyophilized, then analyzed for hydroxy- proline content. The results of the analyses showed that the collagen in the extracts, which had been eluted with 0.2 M NaCl in the first unretained fraction from the DEAE column, contained abundant hydroxyproline. On the other hand, the fraction eluted with 1.0 M NaCl did not contain any hydroxyproline. These results indicate that DEAE- sepharose CL 6B chromatography under the conditions outlined above provided a rapid and efficient method for separating collagen from proteoglycans in cartilage extracts. This is in agreement with the results of Miller (1973). A simple RP-HPLC method was applied to determine purity of type II collagen using a ZORBAX 300 SB column (Fig. 2). Type II collagen was eluted at 10.233 min and the content was 95.4% according to RP-HPLC peak area. The results suggest that the prepared collagen essentially was free from contaminating proteins by the method used in this study for purification. This agreed with the results of Ho, Lin, and Sheu (1997). Sodium sulphate–polyacryl- amide gel electrophoresis (SDS–PAGE) is the most widely used analytical procedure for the identification of collagen chains. Fig. 3 shows the SDS–PAGE electrophoresis pat- terns of type II collagen from the articular cartilage, sternal
Fig. 1. CD spectra of pepsin-solubilized type II collagen treated with various conditions: (a) temperature; (b) time and (c) concentration.
cartilage and reference Sigma–Aldrich. All these collagen samples in purified form had similar migration bands and consisted of an a-chain and their dimmers b-chains with a subunit Mr of 110 kDa. Collagen derived from chick articular cartilage has been characterized with respect to chain composition to comprise of three identical a-chains (Beck & Brodsky, 1998; Helen & Cahir, 2005; Pieper & Van der Kraan, 2002). It may be concluded, therefore, that sternal cartilage collagen is comprised of three identical a- chains with a subunit Mr of 110 kDa like articular carti- lage, and collagen molecule was depicted as [a 1 (II)] 3.
3.3. Biochemical properties of type II collagen
3.3.1. Amino acid composition Bella, Brodsky, and Berman (1995) reported that all col- lagens contained a unique triple–helical conformation as the common structural element; the triple helix consisted of three left-handed polyproline II-like chains supercoiled in a right-hand manner a common axis. The stabilization of this conformation requires the occurrence of glycine res- idues at every third position in the sequence and the pres- ence of a high content of imino acids. Our work detected the composition of amino acid of the chick cartilage to resemble that from the reference Sigma–Aldrich type II col- lagen and articular cartilage in the high content of glycine, hydroxyproline and proline residues, with 310, 117 and 115 residues per 1000 amino acids residues respectively, and small amounts of tyrosine, cysteine, histidine and methio- nine residues with 5, 18 and 10 residues per 1000 amino acids residues respectively (Table 2). Tryptophan residues were absent in all samples because of the hydrolysis of 6 M hydrochloric acid. From these results it can be con- cluded that glycine, which constitutes about 1/3 of all res- idues in collagen, would be present as every third residue in the sequence, and that high amounts of proline and hydroxyproline (Hyp) could be accommodated while main- taining planar peptide bonds. These assumptions led to the construction of the correct model of sternal cartilage colla- gen as a (Gly-X-Y) n pattern, where X and Y are fre- quently Pro or Hyp, respectively. Indeed, it is the most commonly found triplet in collagen chains space (Baum & Brodsky, 1990; Camilla, Karl, Jiann-Jiu, & Christopher, 1996; Nagai & Suzuki, 2000).
Fig. 2. RP-HPLC chromatogram for purified collagen of chick sternal cartilage.
Fig. 3. SDS–PAGE profile for the Sigma–Aldrich standard type II collagen (lane 1), type II collagen purified from chick articular cartilage (lane 2), type II collagen purified from sternal cartilage (lane 4 and 3) and MW standards (lane M).
Table 2 Comparison of amino acid composition of type II collagen from reference Sigma–Aldrich, sternal cartilage and articular cartilage Sigma residues/ residues
Articular collagen residues/ esidues
Sternal collagen residues/ residues Aspartic acid/ asparagine
47 45 46
Glutamic acid/ glutamine
94 88 85
Serine 25 24 22 Histidine 4 4 4 Glycine 313 309 310 Threonine 30 31 26 Alanine 102 99 104 Arginine 53 50 52 Tyrosine 5 4 5 Cysteine 17 18 18 Valine 22 19 19 Methionine 2 8 10 Phenylalanine 15 14 15 Isoleucine 13 10 11 Leucine 31 28 27 Lysine 15 12 14 proline 94 116 115 Hydroxyproline 118 121 117
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