Problems 1

A) The absorbance A of a solution is defined as

A=log(I0/I)
where I0 is the incident light intensity and I is the light intensity after passage through the sample.

Absorbance is related to the molar absorption coefficient (extinction coefficient) (units M-1 cm-1),
concentration c (in M) and path length l (in cm) by
A=lc

The molar absorption coefficient () of myoglobin (Mw 17800 g mol-1) is 15 000 M -1 cm -1 at 580
nm. Which of the following is the absorbance of a 1 mg/ml solution of the protein if the lightpath is
1 cm?
a) 0.00084
b) 15000
c) 0.42
d) 0.84

B) An easy way to determine the critical micellar concentration of detergents or some lipid species is
by following the absorbance (@ 600 nm) of the dye Sudan black when it is added to a lipid aqueous
solution. Sudan black is insoluble in water but is soluble in hydrophobic environments. When lipid
aggregates (micelles or liposomes) are present in the solution, Sudan Black partition into the
hydrophobic core of the lipid aggregate and the presence of the dye can be detected by using a
spectrophotometer. The following table shows the absorbance values obtained in a laboratory for a
detergent X and Y at 25C. The concentration of the detergent stocks solutions is 10 mM for both
detergents, the final volume in each tube is 5 ml:

Volume of X added Volume of Y added Absorbance X Absorbance Y

to the tube (mL) to the tube (mL) (Ax) (Ay)
0.1 0.1 0.010 0.010
0.2 0.2 0.015 0.015
0.3 0.3 0.013 0.013
0.4 0.4 0.012 0.012
0.5 0.5 0.050 0.010
0.6 0.6 0.100 0.015
0.7 0.7 0.140 0.013
0.8 0.8 0.200 0.012
0.9 0.9 0.260 0.070
1.0 1.0 0.290 0.140
1.1 1.1 0.360 0.220
1.2 1.2 0.390 0.300
1.3 1.3 0.440 0.360

a) Define CMC
b) Determine the CMC for both detergents
c) Calculate the G for both process (detergent A and B)
d) The CMC for detergent Y at 90C is 1,7 mM. Taking into account the Vant Hoff relationship,
calculate the H for the aggregation process [(ln CMC)/(1/T) = H/R].
e) Calculate the entropic contribution for the aggregation of detergent Y at 25C.
f) Discuss the hydrophobic effect.
g) Based in your results and the second law of the thermodynamics indicated the CORRECT option:
1) The aggregation process for B is spontaneous because is exothermic (the enthalpy of the
process is negative).
2) In water, micelle formation is driven by an entropic effect originating from the disruption of
highly dynamic hydrogen bonds among water molecules by the nonpolar solute. This process is
spontaneous despite the fact that assembling surfactant molecules together reduces their entropy.
3) The aggregation of lipid A and B are spontaneous because the G is negative in both cases.
However, the aggregation process for A will occurs faster because the G has a more negative
value respect to that observed for B.
4) The driving mechanism for micellization is the favorable interaction among the hydrocarbon
chains in the interior of micelles. This explains the spontaneity of the process.

C) Phospholipase A2 is an innate cellular enzyme that catalyzes the hydrolysis of a

phosphoglycerophospholipid to its corresponding lyso-glycerophospholipid and fatty acids. This
reaction occurs at the position 2 of the sn-glycerol backbone in the glycerophospholipid molecule.

The activity of this protein was tested using the phospholipid indicated below as a substrate:

Fig. 1

Indicate the correct nomenclature for the substrate (see figure 1 above) and the products for the
PLA2 reaction:

a) 1-stearoyl-2- oleoyl-sn-glycero-3-phosphocholine and cis-9-octadecenoic acid and 1-

octodecanoyl-sn-glycero-3-phosphocholine

b) 1,2-dioleoyl-sn-glycero-3-phosphocholine, oleic acid and 1-(cis-9-octadecenoyl)-sn-

glycero-3-phosphocholine

c) 1-stearoyl-2- (cis-9-octadecenoyl)-sn-glycero-3- phosphoethanolamine and oleic acid

and 18:0 lyso PE

d) 1,2-distearoyl-sn-glycero-3-phosphocholine, stearic acid and 1-octodecanoyl-sn-

glycero-3-phosphocholine
D) Indicate the correct statement:
1) Membranes are structurally and functionally symmetric.
2) The degree of fluidity of a membrane partly depends on the chain-length and degree of
unsaturation of the lipids.
3) Lipid bilayers are co-operative structures held together by covalent bonds among lipids, proteins
and sugars.
4) Lipid bilayers are highly permeable to ions.

E) Estimate the number of phospholipid molecules in a 1.0 m2 region of a phospholipid bilayer

membrane, assuming that one phospholipid molecule occupies 70 2 of the surface (1.0 = 10-4
m). Indicate the correct answer:
1) 1.43 106
2) 5.72 106
3) 4.29 106
4) 2.86 106

F) A phase diagram for a binary phospholipids (DSPC/DMPC, Tm 53 and 24 C, respectively)

mixture is shown in the figure. Based on the phase diagram information indicates the incorrect
answer:

1. The membrane phase state in A is fluid

2. The process from A to B corresponds to a fluid
gel/fluid phase transition.
A 3. In B the membrane phase state corresponds to a
coexistence of gel and fluid phases
4. The fluid phase in B is enriched with DMPC
5. The gel phase in B is enriched with DMPC

B
G) The phase diagram for DMPC/DPPE is presented below:

50

45
Temperature ( C)

40

a
o

b
35

30

c
25

20
0.0 0.2 0.4 0.6 0.8 1.0
X DPPE

Indicate the only two correct answers:

1) At point c the molar fraction of DPPE is 0, 4 in the fluid disordered phase and 0,6 in the gel
phase.
2) At point b the fraction of of fluid disordered phase is 0,6.
3) At point a and bthe molar fraction of DPPE in the fluid disordered phase is the same.
4) At point a the fractions of gel and fluid disordered phase are 0,8 and 0,2 respectively.
5) At point c the fraction of fluid disordered phase is the same than at point b
6) at point a and b the fraction of gel phase is the same.
7) At point a the molar fraction of DPPE is 0,8 in the fluid phase and 0,3 in the gel phase.
8) The molar fraction of DPPE in the gel phase is higher in point a respect to point b.

H) A lipid membrane undergoes a phase transition from gel to fluid phase with a Tm = 24C. The
molar enthalpy of the transition is H = 37 kJ/mol.
Knowing that the change of the entropy of the system is S= H/Tm
1. Predict the change of entropy per lipid molecule
2. Considering the Boltzman approximation (S= kB ln ) predict the augmentation in the
number of microstates of one lipid molecule during the phase transition (kB= 1.38066 x 10-23
J K-1).
3. Based on the chemical structure of a lipid molecule, could you predict which part of the
molecule will be the most obvious available source to contribute with all these microstates?
4. Could you predict the tendency of the process at 20 and 30 C?