Papain (3.4.22.2) is a sulfhydry! Protease enzyme that is isolated from the fruit of Carica papaya, the

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Question:

Papain (3.4.22.2) is a sulfhydry! Protease enzyme that is isolated from the fruit of Carica papaya, the papaya (or paw-paw fruit). It has been used as a meat tenderizer and more recently as an agent for the cleansing of "soft" contact lens, although another protease enzyme subtilisin B is now preferred for these lens

This enzyme (papain) is supplied in tablet form and reconstituted in distilled water to remove protein deposits from the surface of contact lens.Azocoll is used as the substrate for papain in this experiment. Azocoll is insoluble, powdered cowhide to which a red dye has been covalently attached. When the peptide linkages of the collagen of the cowhide are hydrolyzed, the azo dye is released into solution. The rate at which the dye is released is measured spectrophotometrically at the dye absorbance maximum of 520 nm,

Materials and Methods

I tablet of papain -dissolved in 10 ml water gives a papain solution.

Azacoll at 500mg/4ml

4ml cuvettes suitable to use at 520nm

Bench spectrophotometers

Clingfilm

Pipettes and tips to measure in range 20-200ul and 1-5ml

Procedure (Students to work in pairs)

Add 125 μl of azacoll stock to a cuvette, then add 2.5ml of enzyme stock. Cover cuvette with clingfilm. Invert to mix and quickly place in spectrophotometer set to detect absorbance at 520nm.

Monitor progress of reaction by recording every minute for 30 minutes.

Plot progress curves of absorbance against time.

Measure initial rate (Vo) from each progress curve, record these and plot against substrate concentration to obtain Michaelis- Menten plots.

Discussion

Interpret your progress curve plots and the Michaelis- Menten plots. Research what you need to plot in order to obtain reliable estimates of Vmax and Km.

How could you determine the effects of pH and temperature on the reactions.