Studies on the quantification of proteoglycans by the dimethylmethylene blue dye-binding method. Specificity, quantitation in synovial lavage fluid, and automation.

The dimethylmethylene blue (DMMB) dye-binding technique is widely used for the quantification of sulfated glycosaminoglycans (sGAG) and proteoglycans. We conducted further studies on this technique in our laboratory and found that concentrations of DNA and RNA in excess of 20 micrograms/ml interfered negatively with the detection of sGAGs; interference was eliminated by using DNase and RNase. Hyaluronan at 40 micrograms per ml did not interfere with the detection of sGAG. However, because of the higher concentrations of hyaluronan in synovial lavage fluid, it was necessary to treat this fluid with Streptomyces hyaluronidase in order to quantify sGAG. The DMMB assay was automated with a laboratory work station and compared to the standard method.     

Sensitivity of monoclonal antibody, 5-D-4, for the detection of aggrecan,aggrecan fragments,and keratan sulfate

Bovine nasal septum aggrecan and selected proteinase-digested products of aggrecan were evaluated in an inhibition ELISA using the anti-keratan sulfate (KS) monoclonal antibody 5-D-4 (5D4). Undegraded aggrecan was recognized with an IC₅₀ of 0.27 μg/ml. When aggrecan was treated with human stromelysin (SLN), human leukocyte elastase (HLE), or papain, the degradation fragments had different hydrodynamic sizes. Treatment with SLN produced the largest fragments, HLE generated intermediate fragments, and papain the smallest fragments. Whereas degradation of aggrecan by SLN had little effect on recognition of proteoglycan in the ELISA (IC₅₀-0.5 μg/ml), degradation by both HLE and papain significantly decreased the sensitivity for detection of KS epitope (IC₅₀-170 and 215 μg/ml, respectively). In addition, 5D4 detected single chain costal and corneal KS with much less sensitivity (IC₅₀-21 and 469 μg/ml, respectively) than undegraded aggrecan (IC₅₀-0.27 μg/ml).

     

In vivo activity of human recombinant tissue inhibitor of metalloproteinases (TIMP). Activity against human stromelysin in vitro and in the rat pleural cavity

Recombinant human tissue inhibitor of metalloproteinase (rhTIMP) suppressed the ability of native human stromelysin to degrade [3H]transferrin in vitro. Maximum inhibition occurred at molar ratios (TIMP: stromelysin) of 2:1 and 1:1. Reduced and alkylated tissue inhibitor of metalloproteinases (TIMP) lost its ability to suppress stromelysin activity. rhTIMP also inhibited stromelysin from degrading proteoglycan monomer in vitro. When injected into the rat pleural cavity prior to stromelysin, rhTIMP inhibited the ability of the enzyme to degrade aggregating cartilage proteoglycan monomer. Marked inhibition of stromelysin-mediated proteoglycan degradation in vivo occurred at molar ratios (TIMP: enzyme) of 2:1 and 1:1, with less inhibition at molar ratios of 0.5:1 and 0.25:1. Reduction and alkylation prevented rhTIMP from suppressing stromelysin-mediated degradation of proteoglycan monomer in vivo. By comparison, an equimolar concentration of the serine proteinase inhibitor, alpha 1-proteinase inhibitor (alpha 1-PI), did not inhibit stromelysin activity in the rat pleural cavity. This study demonstrates that rhTIMP is effective in inhibiting native human stromelysin both in vitro and in vivo.