Purification and characterization of type II collagen

Purification and characterization of type II collagen

(Parte 1 de 3)

Purification and characterization of type I 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 1 September 2007


Type I 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 I 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 I collagen derived from chick cartilage was closer to that of reference Sigma–Aldrich type I collagen which contains more imino acid. Analysis by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR) revealed that type I collagen from chick sternal cartilage retains more intermolecular crosslinks during the purification process. Collagen purified from chick sternal cartilage was typical type I collagen and may find applications in functional foods. 2007 Elsevier Ltd. All rights reserved.

Keywords: Type I collagen; Purification; Sternal cartilage; Characterization; Secondary structure

1. Introduction

Type I collagen is the main structural component of cartilage and, together with other tissue-specific collagens and proteoglycans, provides the tissue with its shockabsorbing properties and its resiliency to stress (Gelse, Poschl, & Aigner, 2003). Type I 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 engineering material, microencapsulation, or tablet coating). Today, there is an increasing demand for type I collagen as research suggests that type I 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 I collagen, traditionally, has been extracted from bovine or porcine articular cartilage. Chick sternal cartilage containing a high amount of collagen, one of the byproducts of chick manufacturing industry is recognized as a potential source of type I 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 I collagen.

Type I collagen has been extracted from articular cartilage. The result indicates that the functional properties of type I collagen are highly influenced by its molecular structure (Miller, 1971; Rigo, Hartmann, & Bairati, 2002). In general, the telopeptide of type I 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 procedure that involved suspending tissue (200 g wet weigh) in

0308-8146/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2007.09.022

* Corresponding author. Tel./fax: +86 510 85884496. E-mail address: syxu@sytu.edu.cn (S.-Y. Xu).


Available online at w.sciencedirect.com

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 preparation of telopeptide-poor collagen by treatment with pepsin (approximate ratio of enzyme to collagen was 1:400) at 20 C with intermittent stirring for 5 days. The main difficulty, 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 I collagen isolated from cartilage. Circular dichroism (CD) is particularly useful for analyzing collagen and associated degradation products, who are able to assign the secondary structure of type I 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 I collagen, with the aim of producing extracted protein with minimal changes to its functional properties. Further, some biochemical characterizations of type I collagen from chick sternal cartilage are also assessed.

2. 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 Phosphorylase b, 97.4 kDa; bovine serum albumin, 6.2 kDa; rabbit actin, 43 kDa) for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) were obtained from Shanghai Huamei Biotech (Shanghai, China). Standard type I collagen and pepsin (EC were purchased 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 chloroform–methanol (2:1, v/v). After the pieces of tissue were cleaned with deionized water, then the chloroform–methanol-free pieces were stored at 20 C until use.

2.3. Preparation of pepsin-solubilized type I collagen

2.3.1. Digestion test

Sternal cartilage was homogenized at 10,0 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 Ca nd 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 I collagen. The resulting viscous 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 dissolved in 0.5 M acetic acid (pH 2.5) and aggregated by dialysis against 0.02 M phosphate buffer (pH 7.4), then lyophilized. The lyophilized collagen was stored in desiccator placed in a refrigerator (4 C), until used.

2.3.2. The yield of pepsin-solubilized type I collagen

The yield of pepsin-solubilized type I collagen with different 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 I collagen

Circular dichroism (CD) spectra were applied to assess the secondary structure of pepsin-solubilized type I collagen from the different digestion conditions. The type I collagen was diluted using 0.05 M acetic acid and then the solution placed into a quartz cell with a path length of 1 m. 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 containing acetic acid was also recorded. The CD spectra of the samples were obtained after subtracting the reference spectrum. The data were accumulated three times.

2.4. Purification of type I collagen

2.4.1. DEAE-sepharose CL 6B ion exchange chromatography

The lyophilized pepsin-solubilized type I 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 dialysis 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-absorbing 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 continued until an additional peak was eluted from the column. The column was reequilibrated with the starting buffer and was ready for reuse.

440 H. Cao, S.-Y. Xu/Food Chemistry 108 (2008) 439–445

2.4.2. NaCl precipitation

The fraction of unretained DEAE-sepharose were pooled and concentrated using 3 M NaCl precipitation. The precipitate was collected by centrifugation, redissolved in 0.5 M acetic acid, dialyzed against a large volume of the same solvent, and then lyophilized.

2.4.3. Polyacrylamide gel electrophoresis

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed for the determination of purity of type I collagen as described by Laemmli (1970), using a 5% (w/v) stacking gel and a 7.5% (w/v) separating gel. The samples were prepared by mixing the purified collagen at a 1:1 (v/v) ratio with distilled water containing 10 mM Tris–HCl pH 6.8, 2.5% SDS, 10% glycerol, 5% b-mercaptoethanol and 0.002% bromophenol blue. Gels were stained with protein Coomassie brilliant blue R250. The molecular weight of the collagen was estimated using a high molecular weight calibration kit as marker.

2.4.4. Reversed-phase high performance liquid chromatography (RP-HPLC)

Prior to HPLC analysis, the purified type I collagen was diluted to 4.5 mg/ml using 0.5 M acetic acid (pH 2.5) and filtered through a 0.25 lm cellulose acetate membrane filter. Chromatography was performed on an Agilent 10 HPLC system (Aglient, Palo Alto, CA, USA) with an ultraviolet detector. The reversed phase column was a 4.6 m 250 m ZORBAX 300 SB (Aglient, Wilmington, DE, USA) with an injection volume of 20 l. The mobile phase consisted of two solvent: (A) 5% acetonitrile and 0.05% trifluoroacetic acid (TFA) and (B) 80% acetonitrile (v/v). The separation was performed using the linear gradient of A–B (v/v). The flow rate was maintained 1 ml/min, and an absorbance was monitored at 220 nm.

2.5. Biochemical properties of type I collagen

2.5.1. Amino acid analysis

For amino acid analysis, the collagen purified from chick sternal cartilages was hydrolyzed with 6 M hydrochloric acid for 24 h at 120 C. The resulting mixture was analyzed by an Agilent 10 HPLC system (Aglient, Palo Alto, C, USA) following online derivatisation with O- phthalaldehyde and 9-fluorenylmethoxycarbonyl (Sigma Chemical Co., St. Louis, MO, USA) for proline.

2.5.2. Denaturation temperature (Td)

The Td was determined by means of a differential scanning calorimetry (Perkin–Elmer Co., Norwalk, CT, USA).

Purified type I Collagen and intact cartilage were immersed in deionized water at 4 C for 16 h. The wet samples were wiped with filter paper to remove excess water and hermetically sealed in aluminum pans. A heating rate of 5 C/min was applied from 20 to 90 C and the endothermic peak of the thermogram was monitored.

2.5.3. Fourier transform spectroscopy (FTIR)

FTIR spectra were obtained from discs containing 2 mg samples in approximately 100 mg potassium bromide (KBr) with a Fourier transform IR instrument (Nicolet Nexus, Thermo Electron Co., Madison, WI). A spectral range of 4000–400 cm 1 (2.5–25 lm) was analyzed and registered in the transmission mode with resolution of 2c m 1.

3. Results and discussion 3.1. Preparation of pepsin-solubilized type I collagen

The pepsin is applied to remove the telopeptide of the collagen because the natural polymer of telopeptide-poor collagen is of low antigenicity, biocompatible, biodegradable, and less toxic. There are several factors affecting the characteristics of telopeptide-poor collagens including the ratio of pepsin to cartilage, temperature, and digestion time (Takaoka et al., 1991). We determined the yield and secondary structure of obtained type I collagen under different extraction conditions, the results are summarized in Tables 1 and Fig. 1.

The temperature, pepsin concentration and times of digestion have a significant effect on the yield of type I collagen (Table 1). The yield for type I collagen obtained, was increased with increase in extraction times, temperature and pepsin concentration. The highest yield 62.5% of type I collagen was obtained with the extraction times for 32 h and 0.5% pepsin concentration at 37 C.

Table 1 Concentration, temperature and times of pepsin digestion effect on the yield and secondary structure of pepsin-solubilized type I collagend

Type I collagen yield (%) Ellipticity (m deg) Secondary structure

Random coil (%)

Concentration (w/v)a 1 60.2a 12.1a 98.7a 4.3a 5.7a 0.5 59.7a 13.0a 103.7a 4.2a 5.8a 0.25 54.7 b 12.8a 9.7a 46.6a 53.4a

Temperature ( C)b 4 53.6a 13.2a 112.1a 46.5a 53.5a 20 58.2a,b 1.9a 95.7a,b 43.4a,b 56.6a 37 62.5b 1.4a 57.2b 39.1b 60.9b

Times (h)c 16 50.7a 13.2a 112.1a 45.6a 54.4a 32 56.1a,b 12.0a,b 97.5a,b 4.1a,b 5.9a 48 58.3b 10.9b 5.3b 39.7b 60.3b a The concentration of pepsin digestion was 0.25%, 0.5% and 1% (w/v). (32 h, 20 C). b The temperature of pepsin digestion was 4 C, 20 C and 37 C, respectively. (0.5% pepsin (w/v), 32 h). c The times of pepsin digestion 16 h, 32 h and 48 h, respectively (0.5% pepsin (w/v), 20 C). d Results are presented as the means (n = 3), where mean within a column followed by different letters are significantly different (P < 0.05).

H. Cao, S.-Y. Xu/Food Chemistry 108 (2008) 439–445 441

CD-spectra of protein solutions provide information about secondary structure content of proteins (Kelly & Price, 1997; Usha, Maheshwan, Dhathathreyan, & Ramasami, 2006). Fig. 1 shows the CD spectra of the pepsin-solubilized type I collagen under different extraction conditions. All collagen samples showed a rotatory maximum at 221 nm (positive band), minimum at 198 nm (negative band) and a consistent cross over point (zero rotation) at about 212 nm, which was characteristic of triple helical conformation of the protein (Usha & Ramasami, 2004). The ellipticity of type I 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, Jefferson, 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.

(Parte 1 de 3)