1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme.

Several gold salts were compared in kaolin-induced rat paw oedema, u.v. erythema in guinea pigs, delayed type hypersensitivity and humoral immunity in mice, and adjuvant-induced arthritis in the rat. In the latter the additional parameters of serum gold and copper levels and lysosomal enzyme activity were determined. In addition, the in vitro inhibition of several lysosomal enzymes derived from mouse macrophages was studied. The gold compounds examined were aurothiomalate, aurothioglucose, triethylphosphine gold chloride (SK & F 36914) and its glucopyranoside derivative (SK & F D-39162), triphenylphosphine gold chloride and sodium gold chloride dihydrate. SK & F 36914 and SK & F D-39162 has significant activity after oral dosage upon paw kaolin and u.v. erythema in rats and guinea pigs, respectively. Gastric swelling also occurred. In Wistar rats, adjuvant arthritis was little affected by the gold salts but in the Lewis rats there was suppression. In both strains there was less elevation in serum copper levels with treatment by SK & F 36914 and SK & F D-39162, but not by aurothiomalate. None of the compounds had any measurable effect on delayed hypersensitivity or humoral antibody levels in mice. The in vitro activities of cathepsin B1 and cathepsin D were inhibited by all the gold compounds. Reactivity of gold compounds with glutathione and cysteine in vitro was dependent on compound solubility and the nature of the gold ligand. Considerable differences exist between the profiles of activity for the different gold salts evaluated. These observations indicate that some gold salts do possess anti-inflammatory activity with a potency similar to that of indomethacin.

Erosion of articular cartilage, which is characteristic of inflammatory joint diseases, including rheumatoid arthritis (RA), is due in part to enzymic breakdown of the components of the cartilage matrix (Bollet, 1963), particularly collagen and chondromucoprotein (CMP). It has been suggested that lysozomal hydrolases, possibly derived from cells of the synovial membrane or from leucocytes present in synovial fluid, are involved in this process (Dingle, 1962;Weissmann, 1966). In this communication we report the existence, in the synovial fluids of patients with rheumatoid arthritis, of protease activity which degrades CMP optimally near neutral pH. It thus differs from the majority of lysozomal enzymes, which are optimally active at pH values lower than those (Goldie and Nachemson, 1969) likely to appertain at the cartilage surface.Material and methods Synovial fluids were aspirated in the course of routine diagnostic or therapeutic arthrocentesis from the knees of patients with a variety of arthritides and the few which were grossly contaminated with blood were discarded. The samples came from 74 patients with RA (68 'definite' or 'classical' and six 'probable'), six with osteoarthrosis, two Charcot joints, one erythema nodosum, one Reiter's syndrome, and three ankylosing spondylitis. A portion of each fluid was transferred to a tube containing ethylene diamine tetra-acetic acid (EDTA) for total and differential cell counting (Cohen, 1967) and the cells were immediately separated from the remaining fluid by centrifuging (15 min.; 1,000, g.; 4°C.). The cells were washed three times with isotonic saline solution, resuspended in a small volume (3-6 ml.) of buffer (0-1 M Tris-HCI, pH 7 * 5) and disrupted by ultrasonication (MSE Ultrasonic Disintegrator, 2 min.; 0-4°C.). Cell debris was removed by centrifuging and the extract tested for CMP-degrading activity. The synovial fluid from which the cells had been removed was also tested.CMP was prepared from bovine nasal cartilage as described by Partridge, Davis, and Adair (1961) (typical analysis: hexosamine 28-0 g./100 g.; uronic acid 25-5 g./100 g.; hydroxyproline 0 20 g./100 g.). Degradation of CMP by cell extracts was detected by measuring the fall of viscosity of solutions in 0 1M Tris-HCI buffer at 25°C. in 2-mi. Ostwald viscometers. A unit of enzyme activity was defined as that amount of enzyme which caused a 20 per cent. fall in specific viscosity in 10 min. when added in 0-2 ml. solution to 2 ml. CMP solution (0 4 per cent.) at pH 7 5; 25°C.Disc electrophoresis of CMP, its degradation products, and chondroitin sulphate (ex shark cartilage) was carried out on polyacrylamide gels by the method of Davis (1964) (spacer gel 5 per cent. acrylamide; pH 8-9; running gel 8 per cent. acrylamide; pH 7 5). The gels were stained for mucopolysaccharide with 1 per cent. Alcian blue in 7 per cent. acetic acid and scanned with a Joyce-Loebl 'Chromoscan' densitometer.Separation of CMP-degrading enzymes in cell extracts was investigated by the disc electrophoresis method of Barre...

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