Measuring Protein Activity (spectroscopy)

Question 1

Why do we measure proteins?

Answer 1

• identity
• concentration
• activity

Question 2

What are destructive ways of measuring protein conc?

Answer 2

• Bradford Assay

- BCA Assay (denatures proteins)

Question 3

What is spectroscopy?

Answer 3

the study of how electromagnetic radiation interacts with matter

Question 4

How does spectroscopy work?

Answer 4

• energy carried by electromagnetic radiation is dependent on the wavelength and frequency
• electromagnetic energy is absorbed by macromolecules

Question 5

What are the types of spectroscopy?

Answer 5

• NMR
• Infrared
• Ultraviolet-Visible

Question 6

When you have a short wavelength do you have a high or low amount of energy?

Answer 6

high

Question 7

What is the plank-einstein equation?

Answer 7

E= hc/ λ
c=f λ
E=hf

E=energy
h=plank constant
c= speed of light
 λ= wavelength
f=frequency

Question 8

How is absorbance measured?

Answer 8

the amount of energy it takes an electron to move from the highest occupied orbital to the lowest unoccupied orbital

Question 9

What does an absorbance of 280nm normally mean?

Answer 9

tryptophan
tyrosine
phenylalanine

Question 10

What does more rings mean?

Answer 10

higher absorbance

Question 11

What absorbance is DNA?

Answer 11

260nm

Question 12

What splits the light into different wavelengths?

Answer 12

prism

Question 13

Where is your sample in spectroscope?

Answer 13

a cuvette after the prism

Question 14

How do you calculate absorbance?

Answer 14

A λ= log10(I0/I)

I0=incident energy
I=transmitted energy

Question 15

What does the absorbance depend on?

Answer 15

• concentration of solute (c)
• light path length (l)
• molar absorption/extinction coefficient

Question 16

What is the path length normally?

Answer 16

1cm

Question 17

What is the Beer-Lambert equation?

Answer 17

A λ= ελcl

ελ= absorbance at 1M^-1cm^-1 solution at wavelength λ of incident energy

Question 18

What is proportional to c?

Answer 18

A λ (measuring absorbance will allow us to calculate the concentration

Question 19

How do you calculate protein concentrations using spectroscopy?

Answer 19

• make a calibration curve using known concentrations

- measure absorbance of unknown compound and use curve to work out conc

Question 20

How can you work out protein concs in an enzyme reaction?

Answer 20

-as reaction goes on, more product is formed and substrate is used up so couple this with absorbance
MUST CONSIDER STOCHIOMETRY BECAUSE SOMETIMES 2 MOLES OF PRODUCT ARE FORMED FROM 1 MOLE OF SUBSTRATE AND VICE VERSA

Question 21

What happens if you can’t use spectroscopy to measure protein conc (no rings)

Answer 21

• link it to something you can see
  e. g a co-factor that changes structure as enzyme works
• link first reaction to another reaction that you can see the products of

Question 22

Why is rate of reaction important when you use coupled reactions?

Answer 22

because if the rate of reaction for the second reaction is slower than the first reaction it won’t truly reflect the rate of reaction of the first reaction (second reaction needs to be going at the same or a faster rate)

Question 23

How can you ensure that the second reaction is going quickly?

Answer 23

use excess enzyme

Question 24

If there are other contaminants present in all of the reactions, how do you measure absorbance?

Answer 24

• add all the absorbances up and compare them fro each different stage
• if you know the amount of absorbance of the contaminants, you know the amount of absorbance fr your compound

Question 25

Is it ok to have contaminants in your reaction?

Answer 25

yes as long as they stay the same