“Science progresses best when observations force us to alter our preconceptions.”
Vera Rubin, Astronomer
What causes the color of traditional collagenase products?
Traditional crude and enriched collagenase products can range in color from a light tan to dark brown. Color is a byproduct of the manufacturing process used to prepare these products. They are derived from Clostridium histolyticum culture supernatants recovered after anaerobic fermentation. To reduce the volume of the fermentation supernatant and to enrich the collagenase activity in the final product, the proteins in the culture supernatant are precipitated with saturated salt solution. The precipitated protein is then resuspended in a smaller volume, and extensively dialyzed to remove low molecular weight contaminants. The final product reflects the critical enzyme activities responsible for tissue dissociation: collagenase and neutral protease activities. In addition to these activities, these products likely contain phospholipase, neuraminidase, hyaluronidase, β-D-galactosidase, β-N-acetyl-D-glucosoaminidase, or α-L-fucosidase activities and variable amounts of endotoxin. The product color reflects contaminating biopigment, a non-descript term for colored proteins.
These enzyme activities will differ depending on the fermentation conditions. The “uniqueness” of each lot led many suppliers to offer lot qualification programs, enabling users to assess the performance of specific lots of product in their cell isolation procedure before purchasing larger amounts of a “good” lot of collagenase product.
The development of purified collagenase-protease enzyme mixtures led to the development of tailored enzyme mixtures designed to recover specific cell populations. The biggest hurdle to overcome was purification of the C. histolyticum collagenase isoforms, class I (C1) and class II (C2), from other enzymes and biopigment. Several column chromatography steps are performed to obtain white, purified collagenases that are > 95% pure. Moreover, endotoxin contamination is significantly reduced. The adjacent infographic shows the steps VitaCyte uses to purify collagenase. The Collagenase HA and MA products are prepared by mixing two purified intermediates purified C1 and C2 in a 60:40 C1:C2 ratio. By contrast, a single column purification step is performed to prepare the Collagenase Gold or Gold Plus products. These products are also > 95% pure but here the C1:C2 ratio is the same as found in the fermentation supernatant: about 75:25 C1:C2 ratio.
The choice you make in selecting a collagenase-protease mixture for enzyme mediated cell isolation is dependent on the degree of control you have on generating consistent results. The table below summarizes the differences in using traditional or PD Collagenases for enzyme mediated cell isolation procedures.
Traditional Collagenase | VitaCyte Collagenase Products | |
---|---|---|
Lot Consistency | Minimal, each lot unique | Maximal: use of purified enzymes ensures consistency |
Collagenase Purity | 4-8% (w/w) for crude 15-25% (w/w) for enriched | > 95% (w/w) |
Need to Pre-Qualify New Collagenase Lots | Necessary to optimize isolation procedure | No need after initial optimization step performed |
The difference in lot consistency reflects the lower purity of traditional collagenase products and the potential of other biochemical components in the product to influence cell viability or function. Moreover, collagenase’s collagen degradation activity can be reduced by proteases present in traditional collagenase products.(1) Van Wart and colleagues identified seven different collagenase forms when they purified collagenase from crude collagenase products.(2) Ten years later, sequencing of the C. histolyticum collagenase genes showed that separate genes coded for C1 (116 kDa) and C2 (114 kDa) proteins.(3) Structure-function studies showed that only those collagenase forms that contain a catalytic and collagen binding domain(s) are “functional” since they can degrade native collagen. C1 had two functional forms (intact 116 kD and truncated 100 kDa) and C2 has one form (intact 114 kDa). This means the four other forms Van Wart purified were non-functional forms of the enzyme: they could cut collagen specific substrates or degrade gelatin but they were unable to degrade native collagen.
VitaCyte’s Collagenase HA, Collagenase Gold, and Collagenase Gold Plus products contain intact C1 and C2 enzymes. The use of a robust purification processes that minimize proteolytic degradation of collagenase at any step of the manufacturing process ensures that the product you buy will have maximal collagen degrading activity (CDA). Collagenase MA is a mixture of intact C1, truncated C1, and intact C2. The specific CDA (CDA U/mg protein) is about 40% of the value of the products note above. This product was developed to replicate the collagenase forms found in several Sigma collagenase products.
Ultimately the choice of the collagenase products you use for your cell isolation procedure depends on your focus as summarized in the table below.
Traditional Collagenase | VitaCyte Collagenase Products | |
---|---|---|
Lifetime Costs for Use | High: costs dependent of availability of good lots | Low: pre-qualify once then done, step eliminated |
Risk to Research Program | If the new lot does match prior results, what to do? | No problem, defined enzyme composition |
Transferability | Replication results by other labs Aligned with translational science | No problem, enzyme composition defined |
Additional Improvements | Not possible | Always an option |
References
- McCarthy RC, Green ML, Dwulet FE. Evolution of enzyme requirements for human islet isolation. OBM Transplantation. 2018; 2(4):[1-30 pp.]. Available from: http://www.lidsen.com/journals/transplantation/transplantation-02-04-024.
- Mookhtiar KA, Van Wart HE. Clostridium histolyticum collagenases: a new look at some old enzymes. Matrix Suppl. 1992;1:116-26.
- Matsushita O, Okabe A. Clostridial hydrolytic enzymes degrading extracellular components. Toxicon. 2001;39:1769-80.