Measuring Collagen-Linked Fluorescence and Browning

1.    Digest 1–5 mg of tendon in 2.5 units/ml of papain (from Papaya latex, Sigma) dissolved in 300  µl of papain buffer

a.     50 mM phosphate buffer (pH 6.5)

b.    2 mM L-cysteine

c.     2 mM EDTA

2.    Incubate 2 h at 65 °C

3.    Measure fluorescence of the papain digests was measured at lex = 370 nm and lem = 440 nm

4.    Measure browning as absorption at 340 nm

5.    Determine hydroxyproline from an aliquot of the papain digest

a.    Hydrolyze in 1 ml of 6 M HCl at 110 °C for 20–24 h

b.    Continue to determine hydroxyproline

Preparation of type I and III collagen standards.          Dorsal skin of a young (80 g) male rat was shaved, delicately cleaned of hair residues with a sharp blade and subcutaneous fat was washed with ether. The skin was minced with scissors into small pieces, 1 × 1 mm each. Skin pieces (1 g) were placed in 200 mL of 0.15 mol/L NaCl in 0.05 mol/L Tris-HCl, buffer (pH 7.4), at 4°C for 12 h with constant shaking at 200 strokes/min. Washed skin pieces were collected by centrifugation at 2500 ×  gfor 5 min at 4°C, and placed in 200 mL of ice-cold 0.5 mol/L acetic acid for 24 h at 4°C with constant shaking at 200 strokes/min. The mixture was finely homogenized and pepsin (1 g/L) was added. Pepsin digestion lasted 16 h at 4°C with constant shaking at 200 strokes/min. The digested homogenate was centrifuged at 30000 ×  gfor 60 min at 4°C, the supernatant collected, and the pellet was redigested and centrifuged under the same conditions. The two supernatant fractions were combined, and collagen was precipitated by adding solid NaCl to a final concentration of 100 g/L. After a 2-h stirring interval at 4°C, collagen was collected by centrifugation at 30000 ×  gfor 30 min at 4°C. Collagen type-III was separated from collagen type-I by precipitation at 1.5 mol/L NaCl as follows: 1) the pellet was completely dissolved in 0.05 mol/L Tris-HCl, 1 mol/L NaCl buffer (pH 7.5) at 4 °C to give a final collagen concentration of 0.5 g/L. 2) The salt concentration was slowly raised to 1.5 mol/L by adding ice-cold 5 mol/L NaCl solution with constant vigorous stirring. 3) The solution was placed at 4°C for 24 h to facilitate maximal collagen type-III sedimentation. 4) Collagen type-III was collected by centrifugation at 40000 ×  gfor 60 min at 4°C. Collagen type-III pellet was treated twice more (steps 1 to 4) to eliminate collagen type-I residues. Collagen type-I in the combined supernatants was collected as follows: 5) the salt concentration was raised to 2.5 mol/L by adding ice-cold 5 mol/L NaCl with constant vigorous stirring, and 6) the solution was placed at 4°C for 24 h to facilitate maximal collagen type-I sedimentation. 7) Collagen type-I was collected by centrifugation at 30000 ×  gfor 30 min at 4°C. The pellet was redissolved in 0.05 mol/L Tris-HCl, 1 mol/L NaCl buffer (pH 7.5) at 4 °C, and was treated once more (steps 5 to 7). Both collagen type-I and type-III pellets were dissolved in 50 mL of 0.5 mol/L acetic acid, dialyzed for 96 h against 0.5 mol/L acetic acid at 4°C with four changes, lyophilized and stored at 80°C.

Analysis of collagen-linked fluorescence.          Bones were lyophilized and crushed to a fine powder. Bone powder (20 mg) was suspended in 1.5 mL of 0.05 mol/L EDTA at 4°C for 72 h with three changes to remove calcium. Decalcified bone powder was washed three times with double distilled water to remove EDTA. Washed bone powder was incubated in 1.5 mL of 0.02 mol/L HEPES buffer (pH 7.5) containing 0.1 mol/L CaCl 2, 350 units of bacterial collagenase type IA (Sigma Chemical) for 24 h at 37°C with mild shaking. Chloroform (2 µL) and toluene (2 µL) were added to prevent microbial growth. A blank containing collagenase in digestive buffer was included. Samples were centrifuged at 13000 ×  gfor 5 min, and the clear supernatant was used for hydroxyproline and collagen-linked fluorescence analysis. Fluorescence measurements were obtained using a LS 50B Perkin-Elmer fluorescence spectrophotometer. Fluorescence was assayed at two distinct wavelengths: 1) emission at 440 nm upon excitation at 370 nm, and 2) emission at 385 nm upon excitation at 335 nm. All fluorescence values were corrected for fluorescence intensity of the collagenase-containing blank solution. Fluorescence data are expressed as relative units of fluorescence per milligram of collagen, with the assumption of hydroxyproline content of 14%.