Azocoll

Gossypol (dimeric sesquiterpene) Gossypium hirsutum, Montezuma speciosissima (Malvaceae) Acrosin, Azocoll proteinase [110, 111]... 

Studies on the activity of rabbit sperm acrosomal enzymes have indicated that gossypol at 12-76 pM could significantly inactivate azocoll proteinase, acrosin, neuraminidase, and arylsulfatase. Hyaluronidase, p-glucuronidase, and acid phosphatase were also inhibited at a higher concentration of gossypol (380 pM) (Yuan et al., 1995). Since acrosomal enzymes play important roles in the fertilization process, the inhibition of... 

Protease Assay. Nonspecific protease activity in largomycin F-II was determined by Azocoll assay. Varying amounts of largomycin F-II (20 yg v 100/yg) dissolved in 20 mM Tris-HCl (pH 7.2) were taken in stoppered tubes. The same buffer was added in each tube to make the volume to 100 yl. To each tube 100 yl of azocoll (100 mg/5 ml water) was added and incubated at 37°C for 1 hour. 

Phosphatase /3-D-glucosidase Proteinase p-nitrophenyl phosphate p-nitrophenyl-/3-D-glucoside Azocoll (protein-dye-complex)... 

Proteolytic activity measurements were made against hemoglobin at pH 2 (16) and Azocoll at pH 5.3 (17). 

Figure 2. Interaction of pepsinogen with pepstatin. The activity of pepsin (0.45 mg) against Azocoll at pH 5.3 was measured in presence ( ) and absence ( ) of pep-...

Insoluble material. This was completely inactive against hemoglobin at pH 2 and Azocoll at pH 5.3. NH2-terminal analysis with dansyl chloride showed both isoleucine and leucine gel electrophoresis at pH 8.1 revealed two bands, one with a mobility similar to that of pepsinogen and the other major band with a mobility intermediate to those of pepsinogen and pepsin. The composition (Table III) is similar to that calculated from the sequence of residues 17-370 in porcine pepsinogen. 

When the pepstatin/pepsinogen molar ratio was lowered to 0.9/1, this insoluble protein fraction had 10% of the specific activity of pepsin against hemoglobin and Azocoll (Table III). 

Activities of soil proteinases have been determined by measuring spectrophotometrically the release of a soluble red azo dye from Azocoll, an insoluble protein containing adsorbed dye and marketed as a substrate for a range of proteinases. The assay required that unshaKen suspensions of soil in phosphate buffer, pH 7, were incubated with substrate for 5h at 37°C. The kinetics of the hydrolysis were not established. Autoclaved soils were inactive. Soil proteinases were increasingly inhibited by HgCl2 in final concentrations ranging from 50 to 1000 pg ml... 

A simple experiment in which these parameters can be obtained by conventional experimental methods uses papain as the enzyme and Azocoll as the substrate. Papain is a sulph-hydryl protease that can be isolated from the fruit papaya (paw-paw), and which is used for tenderising meat. Azocoll is insoluble ground cowhide to which a red dye has been covalently bonded. When the peptide bonds of the collagen are hydrolysed by the papain, the dye is liberated into the solntion and the increase in its concentration as a function of time can be studied spectrophotometrically at 520 nm. 

Experiment 3.6. Experimental determination of the kinetics of hydrolysis of Azocoll by papain [11]... 

Time evolution of the absorbance (A) at 520 nm for various initial concentrations of Azocoll... 

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