Collagen sample preparation

Levels of LWl were quantified by LC/MS/MS in insoluble collagen samples prepared from human donor skin samples obtained by autopsy. The results showed that total LW fluorescence strongly correlated with LWl MS signals at m/z 623 m/z 447 (r=0.94, n=14, P<0.0001). LWl expressed as nmol/mg collagen significantly (P<0.0001) increased with age at an exponential rate in nondiabetic individuals (r=0.92, n=13, P<0.0001). Levels were also significantly elevated by diabetes (P<0.0001), chronic/ESRD (P=0.021), and in non-diabetic patients with severe respiratory problems (P<0.05) . ... 

The limited solubility of membrane proteins and related polypeptides in aqueous mobile phases can also cause problems. These could be solved, e.g., by adding guanidine hydrochloride (6 M) or urea (8 M) to the portion of initial eluent used for sample preparation 69). The urea was always eluted in the breakthrough volume of the column. Thus, the retained hydrophobic polypeptides might have been temporarily precipitated upon the column. Collagen chains, dissolved in 0.5 M acetic acid, were successfully separated by RP-HPLC through gradients of 0.1 M TFA/acetonitrile 70> or (0.05 M ammonium bicarbonate + TFA)/ tetrahydrofuran 57>. 

Figure 5. Internal reflection IR spectra of the surface zones of films of a highly purified rat skin collagen sample (top) ana a more heterogeneous collagen preparation from bovine Achilles tendon collagen (bottom)...

Figure 3. Separations using optimized conditions for Mixture I separation. Eluent B = eluent titrated to pH 6.80 with NaOH eluent C = acetonitrile. The gradient profile is given in Table 1. The column temperature was 34°C. All other conditions were identical to those in Figure 2B. (A) Mixture I standard, (B) collagen hydrolysate sample, prepared as described in the methods section. Total run time was 54 min.

Figure 2 demonstrates the effects of adding octane sulfonic acid to the injection solvent for a reverse-phase separation of pyridinium and deoxypyri-dinium, components of collagen, in rat urine. These polyamine containing compounds are protonated and poorly retained under the usual acidic or neutral mobile-phase conditions. The sample preparation method is simple dilution and does not afford the removal of salts from the sample. Therefore if the analytes were inadequately retained the sensitivity, as well as the method accuracy, would suffer. As the concentration of octane sulfonic acid is increased to 50 mM (Fig. 2a), both the retention time and the peak response for the analytes improve significantly. Litde improvement is obtained at higher concentrations of octane sulfonic acid and retention is not strongly dependent on the injection volume (Fig. 2b). To protect the ion source from the fouling effects of octane sulfonic acid in the injection solvent, a timed divert valve was inserted before the ion source to shunt the excess ion pair reagents to waste during the first few minutes of each injection.

The film prepared at 50 C differs in that it no longer contains the triple helix structure. If the helix-coil (or collagen gelatin) transformation is complete, the absorption should no longer be related to the different structural levels but instead controlled by the tortuosity of the diffusion paths. The rate of water absorption is considerably slower with a decrease in the weight absorbed at low humidities. But the energy profile remains similar to the sample prepared at 20 C. We believe that the disappearance of the triple helix structure is incomplete and that the water tends initially to be absorbed by the structures still present. 

A specific heavy-metals contamination (chromium) was detected in collagen hydrolysates prepared from trimming/cuttings of chromium-tanned hides up to 100 ppm of chromium was found in samples of powdered collagen hydrolysates on the market at the end of the 1970s (135). The oxidation state of chromium was not determined, but probably it was Cr ", less toxic than Cr, as in the tanning industry only basic salts of Cr " are used. 

Studies of the enzyme content of cells frequently involve the use of coarse tissue samples of either animal or plant origin. In such cases some preliminary dissection of the tissue may be necessary to isolate the relevant tissue components and remove unwanted structural material such as collagen, cellulose, etc., before moving on to the more critical disruption of the cells. Sometimes it is possible to use the technique of tissue culture to provide pure cell preparations for subsequent studies. 

In contrast to soft biologies, whose mechanical properties primarily depend upon the orientation of collagen fibers, the mechanical properties of mineralized tissues, or hard biologies, are more complicated. Factors such as density, mineral content, fat content, water content, and sample preservation and preparation play important roles in mechanical property determination. Specimen orientation also plays a key role, since most hard biologies such as bone are composite structures. For the most part, we will concentrate on the average properties of these materials and will relate these values to those of important, man-made replacement materials. 

Figure 4. Sorption of p-galactosidase by collagen preparations (samples as in Figure 2) at different degrees of lysine content as a double reciprocal plot for control, Ac — 14 X 10 6 for 15% modification, A0 — 0.53 X 10 6 for 30% modification, Ac = 0.41 X 10 6 for upper curve, A = 0.18 X 10 6 mol/g collagen,...

Any study of collagen by similar procedures is hampered by several drawbacks. The most difficult problem is that of purity of the original protein. With insoluble collagen, especially, it is very difficult to prepare a sample free from impurities. As the impurities include mucoproteins and elastin, which have carbohydrate components, it will be essential to avoid confusion with these possible contaminants. 

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