Principles of Biochemistry (2+1)

Estimation of Lysozyme activity

• Dilute required amount of reconstituted standard lysozyme 1:3 i.e. (0.3 ml of standard lysozyme + 0.6 ml of phosphate buffer) to bring down the concentration to 0.33 mg/ml (from 1 mg/ml to 0.33 mg/ml) Note: Store the remaining lysozyme at 4ºC and use as standard for two more experiments.
• Dilute the crude sample and eluted sample to 0.33 mg/ml based on the protein concentration is 1 mg/ml, dilute it three times with phosphate buffer to bring down the concentration to 0.33 mg/ml
• Zero the spectrophotometer against phosphate buffer blank
• Dilute required amount of MLU in phosphate buffer such that A280 is between 0.5 and 0.7 Note: Store the remaining reconstituted MLU-full form at – 20ºC and us as substrate for one more experiment.
• Take 3 ml of diluted Mlu substrate in a cuvette and measure the absorbance at A450 against phosphate buffer blank, this is A450 at ‘0’ second
• To the substrate, add 50 µl of standard lysozyme and note the time
• Mix the contents of cuvette for 15 seconds
• Measure the absorbance exactly after 60 seconds of addition of lysozyme
• Repeat steps 5 to 8 for the crude and eluted samples
• Note down the readings as in Table 1.

Time	A280
	Standard	Crude	Eluate
0 Sec.
60 sec.
∆A450