Introduction
Type II collagen is an important structural protein that is essential for maintaining the strength and flexibility of cartilage and other connective tissues where it is mainly found. Recently, un-denatured (native) type II collagen has been used as a dietary supplement to support joint health (1). Many companies use chicken cartilage as a raw material, but fish cartilage can also be a possible alternative. This study focuses on the detection of type II collagen solubilized from salmon cartilage using a commercially available type II collagen ELISA kit (Cat# 6018, Chondrex, Inc.).
Procedures
Salmon cartilage was homogenized in distilled water. The homogenate was then treated with 6M guanidine at 4°C for 16 hours. After centrifugation at 10,000 rpm for 20 minutes, the pellets were washed five times with distilled water by repeat centrifugation.
The washed pellet was suspended in 0.05M acetic acid at 4°C overnight. The resulting suspension was centrifuged at 10,000 rpm for 20 minutes, and the supernatant was collected as the acid extracted sample. The pellet was digested with 1 mg/ml pepsin in 0.05M acetic acid at 4°C overnight. The digested sample was centrifuged at 10,000 rpm for 20 minutes, and the supernatant was collected as the pepsin extracted sample. This pepsin digestion was repeated two more times (for a total of three times). All three supernatants were combined and neutralized to pH 7.5 with a final concentration of 0.1M Tris buffer pH 7.5 containing 0.2M NaCl. The pellet was suspended in 0.1M Tris-buffered saline (TBS) at pH 7.5 at 4°C overnight. The suspension was centrifuged at 10,000 rpm for 20 minutes, and the supernatant was collected as the TBS extracted sample. The pellet was saved and solubilized in a SDS-sample buffer.
For protein separation and visualization, a 6% SDS-PAGE was performed under reducing conditions. The pepsin-and TBS-extracted samples were combined, then dialyzed against 0.05M acetic acid and lyophilized. This lyophilized sample is the salmon type II collagen fraction. The collagen concentration was measured using a hydroxyproline assay kit (Cat# 6017, Chondrex, Inc.). The cross-reactivity of salmon type II collagen with chick type II collagen was evaluated using a type II collagen ELISA kit (Cat# 6018, Chondrex, Inc.).
Results
Gel Electrophoresis
Figure 1 shows the protein profiles of the fractions at different stages of collagen solubilization. The acid extraction with 0.05M acetic acid did not yield any detectable proteins. In contrast, pepsin digestion successfully solubilized proteins, indicating its effectiveness in solubilizing collagen. The protein profile obtained after extraction with 0.1 M TBS at pH 7.5 was similar to that of the pepsin-solubilized fraction, suggesting the presence of soluble collagen components. Based on the band patterns, these samples predominantly contain type II collagen with no type I collagen present. Insoluble samples solubilized by SDS-sample buffer exhibited a distinct protein pattern.
Figure 1. A 6% gel electrophoresis of collagen solutions under reducing conditions, CI: Chick type I collagen, CII: Chick type II collagen, Acid: Acid extracted sample, Pepsin: Pepsin solubilized sample, TBS: Tris buffer extracted sample, and Pellet: insoluble sample.
Hydroxyproline Assay
The salmon type II collagen fraction samples were assayed using Chondrex, Inc.'s Hydroxyproline Assay Kit (Cat# 6017) to determine collagen concentration. Hydroxyproline (HP) has a unique distribution in tissues and proteins, being highly specific to collagen in the extracellular matrix. On average, HP constitutes about 13.5% of collagen's amino acid sequence. The salmon type II collagen fraction was analyzed using a hydroxyproline assay to determine collagen content. The fraction contained a significant amount of collagen, with 97 μg of collagen per 1 mg of sample by weight (9.7%).
Type II Collagen ELISA
The salmon type II collagen fraction samples were assayed using Chondrex, Inc.'s Type II Collagen ELISA Kit (Cat# 6018) to evaluate the cross-reactivity rate (Figure 2). The kit exhibited an average reactivity rate of 71% for salmon type II collagen compared to the chicken type II collagen standard. This suggests that while the kit can detect salmon type II collagen, its reactivity differs from that of the chicken standard.
Figure 2. Reactivities of chick and salmon type II collagen in a type II collagen detection kit
Conclusion
The study confirms that Chondrex, Inc.'s Type II Collagen ELISA Kit (Cat# 6018) can effectively detect salmon type II collagen with a 71% reactivity rate compared to the chicken type II collagen standard. This indicates some variability in detection based on the source of type II collagen. To obtain a more accurate measurement of native salmon type II collagen content, it is recommended to adjust the ELISA results by multiplying them by a correction factor of 100/71. This adjustment compensates for the observed reactivity differences and provides a more accurate estimate of collagen content in the samples.
Reference