2. If a myoglobin solution has an OD409 value of 1, what percentage of light doe

Question

2. If a myoglobin solution has an OD409 value of 1, what percentage of light does it transmit at 409nm? What percentage of light does it absorb at 409nm? (20 pts) Why do you have to mix guanidine with water first before you add the protein? What will happen if you mix protein with undiluted denaturant first then add water? (20 pts) 3. Calculate the volume of each component for each assay and copy the values into your lab manual to use in the lab. (20 pts) 4. guanidine concentration 0.3 0.6 0.9 1.2 5 1.8 2.1 24 2.7 3.0 Myoglobirn 1 mg/ml Guanidine Stock (6 M) Water After let your 50 and 65 samples sit at room temperature, how do you think the absorbance will change? Can you explain why? (20 pts) 5.

Explanation / Answer

Answer 2:- In spectrophotometer or colorimeter, absorbance of the test compound is measured against the blank reference. This absorbance is expressed as optical density (OD). OD measured for a test compound against blank at particular wavelength for e.g. 409nm is OD409. The Absorbance can range from 0% (100% transmission) to 100% (0% transmission). Generally OD is represented as log of the ratio of % absorbance of blank to the % absorbance of test sample.

Thus, Absorbance (Optical Density) =log10 Io/I,

Where, Io = % transmission of blank and I = % transmission of test sample.

Thus OD409= 1 means,

1 = log10 Io/I,

Now % transmission of blank = 100%.

Antilog 1 = 100/x

10 = 100/x

X = 100/10

Thus, x = 10. That means % transmission of test sample = 10%.

Therefore, % absorbance of test sample = 100-10 = 90%.

Thus, OD409 = 1 means 90% of the light is absorbed by myoglobin solution and 10% of light is transmitted by myoglobin solution.

Answer 2:- Proteins tend to aggregate when added in water when present in higher concentration. Guanidine tends to dissolve in water and acts as a stabilizer for protein. The protein molecules will not aggregate when guanidine is present in the water. Thus, guanidine avoids aggregation of proteins in water and thus helps solubilizing the proteins.

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