Elastolytic Activity by Spectrophotometry

PROCEDURE FOR DETERMINATION OF ELASTOLYTIC ACTIVITY BY SPECTROPHOTOMETRY

The following procedure is an example using Elastin-Rhodamine, 200-400 mesh. Other substrates may likewise be used. Each substrate has a distinct absorbency after being solubilized by elastase. The absorbance of the soluble peptides of dyed elastins follows;

Elastin-Rhodamine, 550 nm
Elastin-Fluorescein, 495 nm
Elastin-Orcein, 570 nm
Elastin-Congo Red, 485 nm
Elastin-Remazol, 595 nm

The procedure closely follows published methods (9,10,11,35,36).

Step 1. Known quantities of substrate are totally solubilized by elastase. The optical density per mg of substrate is determined.

Step 2. Known quantities of elastase are incubated with substrate. The quantity of substrate solubilized per mg of elastase (i.e., specific activity) is determined. The elastolytic activity of an unknown can be determined in comparison to the activity of elastase.

Materials required


1. Substrate Suspension; 20mg/ml in 0.2 M Tris-HCl pH 8.8.
2. Buffer; 0.2 M Tris pH 8.8.
3. Elastase; 2X crystallized or chromatographically purified.
4. Conical Flask, 10-25 ml size or Test Tubes.
5. Dubnoff type incubator; equilibrated at 37°C.
6. Magnetic stirrer.
7. Ice Bath.
8. Filter paper, Whatman No 41 or equivalent.

STEP 1. DETERMINATION OF OPTICAL DENSITY OF SOLUBILIZED SUBSTRATE

Examine the following protocol and pipet buffer then elastase into 10 ml Flasks. Keeping the substrate in suspension by stirring magnetically, add aliquots to the flasks using a 1.0 ml blow-out pipet. NOTE> The elastase aliquot should contain 20-30 units activity. This is an excess amount which is necessary completely solubilize the substrate within 30-60 minutes.
 

          Observed O.D. per mg
Flask Buffer Elastase Substrate   O.D. Substrate (Calculated)
(No.) (ml) (ml) (ml) (mg) -------- --------
1(blank) 2.90 0.10 0 0 -------- --------
2 2.65 0.10 0.25 5.0 -------- --------
3 2.40 0.10 0.50 10.0 -------- --------
4 2.15 0.10 0.75 15.0 -------- --------
5 1.90 0.10 1.00 20.0 -------- --------


Stopper the flasks and incubate at 37° with 40-60 excursions per minute until all of the substrate has been solubilized (30 – 60 minutes). Bring the volume to 10 ml with buffer and read the O.D. in 10 mm cuvettes against the blank. A secondary dilution must be prepared in order that all readings fall within the range of the spectrophotometer. Record each O.D. per mg (mulitiplied times the dilution factor) and calculate the O.D. per mg of substrate. The O.D. per mg should be nearly constant (±5%). A plot of O.D. vs mg of substrate should be a straight line.

STEP 2. DETERMINATION OF UNITS ELASTOLYTIC ACTIVITY

Examine the following protocol and pipet buffer then elastase then substrate into 10 ml flasks. Keep the substrate in suspension when delivering aliquots. The elastase concentration should be 0.2 mg per ml in buffer.
Extinction Coefficient: Î, 1%, 280 nm = 19.5 A280x 0.51 = mg/ml

          Observed O.D. per mg
Flask Buffer Elastase Substrate   O.D. Substrate (Calculated)
(No.) (ml) (ml) (ml) (mg) 0 0
1(blank) 2.0 0 0 1.0 -------- --------
2 1.9 0.1 0.02 1.0 -------- --------
3 1.8 0.2 0.04 1.0 -------- --------
4 1.7 0.3 0.06 1.0 -------- --------
5 1.6 0.4 0.08 1.0 -------- --------
6 1.5 0.5 0.10 1.0 -------- --------


Stopper the flasks and incubate with shaking at 37°C for 20 minutes. Submerge the flasks in ice and bring the volume to 10 ml with cold buffer. Rapidly filter through No. 41 paper into cuvettes and read the O.D. of the filtrates against the blank. Record observed O.D. Calculate mg of substrate solubilized by dividing observed O.D. by the constant O.D. per mg previously determined in Step 1.

Observed O.D.

Units = mg Substrate solubilized = O.D./ mg substrate
mg                   mg Elastase                     mg Elastase

Calculate mg substrate solubilized per mg of elastase. This final calculation gives specific activity. The most widely used unit definition for specific elastolytic activity is: One unit will solublized 1 mg of elastin in 20 minutes at pH 8.8 and 37°C.

The elastolytic activity of an unknown may be determined by substituting aliquots of the unknown for elastase in the above procedure. The relative amounts of buffer and unknown can be varied; however, the final volume of the incubate (3 ml), the pH (8.8) and the molarity (0.2 M Tris) must be constant.

DETERMINATION OF ELASTOLYTIC ACTIVITY BY FLUOROMETRY


The same procedures (above) are used for the determination of fluorescent intensity of solubilized substrate and for determinaation of units elastolytic activity. Either Elastin Fluorescein of Elastin Rhodamine are suitable substrates. Usually the filtrates must be diluted with buffer so that the fluorescent intensity reading fall within the read-out capacity of the fluorometer.

When using the Elastin Fluorescein as substrate the fluorescent intensity is read with the excitation and emission wavelengths set at 490nm and 520nm, respectively. When using Elastin Rhodamine the excitation and emission settins are 550nm and 570 nm, respectively.