Peripheral rheumatoid ulceration and evidence for conjunctival collagenase production.

Two patients with rheumatoid arthritis and peripheral corneal ulcerations were successfully treated by conjunctival resection. The tissues removed were assayed by a variation of the radial diffusion method for tissue collagenases. We used an agarose matrix containing lathyritic rat skin collagen. Wells 3 mm deep were punched in the agarose-collagen and surgical specimens were placed in the wells. Spaces remaining in the wells were filled with balanced salt solution. The assay dishes were incubated for four days at 32 degrees C near 100% humidity. Under these conditions release of collagenase was detected by the clearing of diffuse zones in the gel surrounding the well. Conjunctiva proximal to the ulcer produced definite zones of lysis, whereas control specimens taken remote to the ulceration produced no lysis. This direct evidence for collagenase involvement offers an exploration for the beneficial effects of conjunctival resection.

Mercaptoimidazolylpropionic acid hydrobromide. Inhibition of tadpole collagenase and related properties.

A mercapto analogue of histidine (1), (RS)-2-mercapto-3-(5-imidazolyl)propionic acid (2), was prepared by treatment of (RS)-2-bromo-3-(5-imidazolyl)propionic acid with trithiocarbonate. Decomposition of the resulting intermediate with hydrochloric acid followed by Sephadex G-15 chromatography permitted isolation of 2 as a hydrobromide complex having unusual stability and properties as evidenced by IR and 1H NMR data. The potency of this complex in inhibiting tissue (Rana catesbiana) collagenase was estimated by radial diffusion assay. The amount of 2 required to produce 50% inhibition was 3.8 +/- 1.5 mM compared to 8.7 +/- 2.5 mM for cysteine. Preliminary tests of oxygen susceptibility, mutagenicity, and toxicity suggest that this substance may warrant study as a therapeutic agent for control of collagenase-linked corneal ulcerations.

Radial diffusion assay of tissue collagenase and its application in evaluation of collagenase inhibitors.

A radial diffusion assay for tissue collagenase (EC 3.4.24.3) has been devised which is simple, sensitive and capable of application to large numbers of samples. The assay employs an agarose matrix containing solubilized lathyritic rat skin collagen as substrate. Fibril formation is induced for 2 h at 37 degrees C subsequent to 41 h digestion at 28 degrees C. The procedure results in sharply defined zones of lysis which may be measured directly or after photography. The characteristics of the procedure are otherwise similar to those reported for other radial diffusion assays. The new method was used to examine the action of 10 compounds which were known or potential inhibitors of tadpole collagenase. The concentration of inhibitor required to produce 50% inhibition is reported for the following compounds: alpha2-macroglobulin, 142 microng/ml; N-acetylcysteine, greater than or equal to 100 mM; cysteine, 8.7 mM; EDTA, 0.46 mM; histidine, greater than or equal to 100 mM; 2,3-dimercaptopropanol, 0.5 mM and mercaptoacetic acid, 70 mM. The procedure also has potential for clinical determinations (e.g. tears, synovial fluid) since assay dishes may be prepared in advance and only 15 micronl of sample is required.

The effect of calcium chloride on the activity and inhibition of bacterial collagenase.

The enzymatic behavior and inhibition patterns of collagenase of Clostridium histolyticum in the presence of 0.5 M and 3.4 mM CaCl2 have been examined viscosimetrically. The more concentrated salt was found to enhance the rate of digestion of calfskin collagen when either measured viscosimetrically or colorimetrically by trinitrobenzenesulfonate. However, the rate of digestion of calfskin gelatin is unaffected by 0.5 M CaCl2 as determined colorimetrically. Calcium chloride also proved to have a marked effect on the inhibitory behavior of a series of imidazole compounds. Histidine (10mM) is about three-fold more effective as an inhibitor in 0.5 M CaCl2 than in 3.4 mM CaCl2, whereas a reverse effect is true for histamine, Imidazolylpropionate (10mM) was only weakly inhibitory (16%) in 0.5 M CaCl2 and not at all in 3.4 mM CaCl2. Inhibition by 10 mM imidazole was not detectable. These observations may be useful in the design of inhibitors for tissue collagenases which share a number of common characteristics with the bacterial enzyme.