332 JOURNAL OF COSMETIC SCIENCE A 100% "iii (/) ·e 50% (/) e I- 0%-- B C 0 "iii (/) 100% ·e 50% (/) e I- 400nm 500nm 600nm 2 1 3 o%L_========::============== 2 400 nm 500nm 600nm Wavelength Figure 1. UV/visible spectra for three collagens for cosmetic use: (1) AteloHelogen®, (2) Collasol®, and (3) CLR Collagen® at the concentration supplied (A) and after dilution to equal concentrations of 3 mg/ml with water (B). ELECTROPHORESIS Various electrophoretic techniques are suitable for collagen analyses. These techniques are cheap and readily performed, particularly if commercially available pre-cast gels are used. In the examples, (Figures 3-5), the three different collagens for cosmetic use have been compared. Since the stain that is used to detect the collagenous components in each of the electrophoretic methods is not collagen-specific but stains all proteins, these electrophoretic methods also allow any protein contaminants (serum albumin, for ex­ ample) to be detected in collagen samples. In SDS-PAGE (Figure 3), the molecular weight distribution of the individual chain components after denaturation is shown. The single-chain components, the a-chains, are the fastest moving components from intact collagens. These chains show that Atelo­ Helogen® contains type III collagen, while this is absent from CLR Collagen®. Collasol® shows an atypical chain pattern, which also prevents assessment of any type III collagen present. None of the collagens show any significant bands moving faster than the a-chains if present, these bands could indicate degradation products, although they
COLLAGEN EVALUATION 333 2 %T 4000.-0 1500 )000 cm-) Figure 2. Infra-red spectra for three collagens for cosmetic use: (1) AteloHelogen®, (2) Collasol®, and (3) CLR Collagen®. may also occur if the sample is heated in sample buffer for 2 min prior to electropho­ resis. Crosslinked chain components, the dimer r3- and trimer )'-chains, for example, move more slowly on the gel. These indicate (Figure 3) that AteloHelogen® has fewer crosslinked components than the other collagens. The bands that are present are those normally shown by purified type I and type III collagens (22), and no bands due to other collagen types are evident. Again, the pattern shown by the Collasol® is complex and atypical, with a high proportion of crosslinked material, much of which is too large to enter the gel. Electrophoresis can also be performed under non-denaturing conditions, and uses highly porous gels to allow the larger collagen molecules to migrate (14). This system (Figure 4) shows the soluble collagen that is present in a preparation. Collagen dimers also enter the gel, but insoluble or highly polymeric material does not enter. In the example (Figure 4), it can been seen that all three samples contain soluble monomeric collagen of native size. AteloHelogen® shows fewer crosslinked dimer components than CLR Collagen®. Collasol® shows a poor, streaky pattern, with less collagen behaving as a soluble component. This suggests that much of this collagen may be crosslinked or have been modified during preparation. If the isoelectric point is low (see below), then material may not enter the gel as readily at pH 3.1. Gel permeation chromatography can also be used to examine the molecular weight distribution of soluble material in a collagen preparation (23). However, this requires specialized equipment and columns. Also, as all insoluble material must be removed prior to analysis so that it does not block the flow in the column, the data are not representative of the complete sample. Electrophoresis provides a simpler, better method.
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