MCQ 1

Question 1

what is spectrophotometry

Answer 1

the measurement of light absorption or transmission

Question 2

what can spectrophotometry be used for

Answer 2

protein quantitation/concentration, identification, enzyme activity of enzyme catalysed reactions, DNA quantitation, bacterial cell count, chemical detection of carbohydrates

Question 3

what do nanometer and angstrom equal in meter

Answer 3

nm = 10^-9m, A = 10^-10m

Question 4

what is the formula for speed of light and their units

Answer 4

c (cm.sec^-1) = (wavelength (cm) ) (frequency (sec^-1))

Question 5

what is the formula for wave number and their units

Answer 5

wave no. = 1/wavelength(cm)

Question 6

what are the components of a spectrophotometer

Answer 6

light source, entrance slit, prism, exit slit, sample holder, detector

Question 7

what does the beer lambert law state

Answer 7

absorption of light is directly proportional to the concentration of the absorbing substance and the length of the light beam

Question 8

what does the principle of the beer lambert law enable

Answer 8

the calculation of the concentration of a solution by measuring its absorbance

Question 9

what are the two formulas for absorbance

Answer 9

A = log io (incidental light) /i (transmitted light), and A = (E)(c)(l) E-molar extinction coefficient (M^-1cm^-1) c - concentration (M) l - pathlength (cm, almost always 1cm)

Question 10

what OD is absorbance linear up until

Answer 10

OD 3.0

Question 11

what is the purpose of a blank and what is it

Answer 11

a blank is needed for all absorbance measurements to be made relative to, it contains all components of the assay except the compound being measured (eg. the enzyme in an enzyme-catalyst measurement

Question 12

what are the two techniques for protein quantitation using spectrophotometry

Answer 12

direct - ultraviolet absorption - Abs280nm, and indirect - colorimetric assays Abs540-750nm

Question 13

what are the differences between UV absorption and colorimetric assays

Answer 13

UV called direct as does not require the addition of a reagent, does not need a standard curve, protein is not destructed so further analysis can take place, Abs200-210 and 280nm, quartz cuvettes must be used as plstic absorbs at wavelengths less than 320-340nm

protein must react with a reagent to produce a chromophore, standard curve required, cannot be used for further analysis as has been modified, Abs540nm-biuret, Abs562nm-BCA, Abs590nm-coomassie blue/bradford, Abs600-750nm-lowry

Question 14

what are the two formulas used for direct protein quantitation (instead of a standard curve) and give examples of BSA value for both

Answer 14

A 1%(W/V)280nm = 10.0 BSA = 6.3, or A 1mg/ml 280nm = 1.0 BSA = 0.63

Question 15

what is a limitation of Abs280nm and a fix for it

Answer 15

some detergents and nucleic acids also absorb at this Abs which can cause inaccuracies, fix it by co-measuring at 260nm and 280nm and use a formula to determine the value

Question 16

what is the formula used when co-measuring at 260 and 280nm

Answer 16

protein conc. (mg/ml) = 1.55(Abs280nm) - 0.76(Abs260nm)

Question 17

what is being detected at Abs200-210nm

Answer 17

peptides (small proteins)

Question 18

what are the more commonly used colorimetric assays

Answer 18

bradford or BCA, biuret and lowry relatively redundant as much more cumbersome than the former two named

Question 19

what is the principle of the coomassie/bradford assay

Answer 19

addition of protein to dye in acidic solution which stabilizes the ionic form of the dye by hydrophobic and ionic interactions, the dye mainly reacts with arginine (R) residues as well as others in lesser extent (histidine, lysine, tyrosine, tryptophan, phenylalanine

Question 20

what is the principle of BCA (bichinchonic acid) assay

Answer 20

under alkaline conditions CU^2+ forms a complex with the protein’s peptide bonds and is reduced to CU^+ which BCA then captures and the entire complex is kept stable by the alkaline conditions

Question 21

what was the assay method and reagent used in the colorimetric practical we carried out

Answer 21

Bio-Rad reagent based on bradford assay

Question 22

what is the equation for calculating volume required

Answer 22

V1 = (V2xC2)/C1
V1 is volume required, V2 is final volume, C1 is initial stock concentration, C2 is final concentration required

Question 23

what is activation energy

Answer 23

the amount of energy, in calories, required to bring all the molecules of 1 mole of substance at a given temperature to their activated state

Question 24

how does chemical reaction A to P take place

Answer 24

at any given moment a certain fraction of the A population of molecules must possess much more energy that the remainder of the population so that they can overcome an energy barrier and make or break chemical bonds to lead to the production of P

Question 25

what is the rate of reaction dependent on

Answer 25

the number of A molecules that attain enough energy to pass over the energy barrier

Question 26

what can stimulate a chemical reaction to occur

Answer 26

heating the reactants, catalysts (enzymes)

Question 27

how to catalysts work

Answer 27

catalysts accelerate chemical reactions by lowering the activation energy required

Question 28

every chemical reaction has...

Answer 28

a specific enzyme catalyst

Question 29

why are enzyme catalysed reactions important to the cell

Answer 29

for metabolic regulation where a simple enzyme E forms with its substrate S a complex ES, this complex ES could dissociate to reform enzyme E and substrate S or the complex could break down to form product P

Question 30

what are factors that affect the velocity of enzyme catalysed reactions

Answer 30

concentration of enzyme catalysing the reaction, reaction time, pH of the reaction medium

Question 30

what are the units in which enzyme activity is expressed

Answer 30

katal (symbol is kat) or microkatals

Question 31

what is the formula for converting product concentration into enzyme activity

Answer 31

umoles product formed/incubation time (seconds) = enzyme activity in ukat

Question 32

how to do test to determine the activity of alkaline phosphatase

Answer 32

construction of standard p-nitrophenol curve, effect of incuabtion time on enzyme activity, effect of enzyme concentration on substrate hydrolysis, effect of pH on enzyme activity

Question 33

where is the enzyme alkaline phosphatase found

Answer 33

widley distributed in bone, milk, kidney, intestines, certain microorgansims

Question 34

what is alkaline phosphatase optimum pH

Answer 34

10

Question 35

what is alkaline phosphatase inhibited by

Answer 35

chelating agents and inorganic phosphate, requires Mg^2+ for full activity

Question 36

what is bovine alkaline phosphatase molecular weight

Answer 36

100,000

Question 37

what does alkaline phosphatase hydrolyse and what does it yield

Answer 37

natural and artificial phosphate esters such as p-nitrophenylphosphate which yields an orthophosphate (H3PO4) and an alcohol p-nitrophenol

Question 38

how can p-nitrophenol amount be measured

Answer 38

spectrophotometrically at 405nm or colorimetrically at 430nm

Question 39

what is enzyme polymorphism aka, how can it be seperated and what is analysis used for

Answer 39

multiple molecular/isoensymes, seperated electrophoretically or by ion-exchange on DEAE cellulose, analysis to distinguish between liver lesions and bone lesions

Question 40

what is used to stop the enzymic reaction

Answer 40

0.02M NaOH, doesnt affect the yellow colour of p-nitrophenol

Question 41

at pH 8 what are the roles of 0.02M Naoh

Answer 41

to stop the reaction and to intensify the colour and shift the pH so far from the pK of p-nitrophenol that minor variations in the pH will not significantly affect the colour yield