Erika Mae R.

Sia
S 3L

01/14/2014

Answers to Guide Questions

1. The gelatin inside the artificial membrane will allow the presence of negatively charged proteins
to the cell and thus, causing an electrochemical gradient and restricting anions to enter and
favouring only the entry of cations. Due to its high molecular weight, gelatine is also
impermeable to the dialyzing membrane. Generally, the gelatin granules will enable the inside
portion of the dialyzing membrane to become more negatively charged (net) as compared to
the outside which can be considered neutral (net).
2. The initial pH inside the bag is 4.65 while the final pH is 4.01. This means that the acidity level
inside the bag was reduced by accumulating H+ ions from the outside the dialyzing membrane
via electrochemical and concentration gradient. H+ ions move from high concentration (outside)
to lower concentration (inside) and was attracted by the negatively charged gelatin. On the
other hand, Cl- ions pulled by concentration gradient will be deflected by the electrochemical
gradient since it is already negatively charged. The pH of the outside environment has increased
(more basic) and thus, proves the migration of H+ ions. The migration and repelling reactions is
accounted by Donnan Equilibrium refers to attaining of an electrically neutral environment of
both sides of the membrane.
3. Without pH meter, the use of pH paper and pH indicator dyes can be used. pH paper changes
according to the basicity and acidity level of a solution. Blue if basic and red if acidic. For pH
indicator dyes, they will be added to the solution being adjusted. For example, if a solution
needs to be acidic, phenylalanine can be added prior to addition of acid. A pink color will flash
when the solution is already acidic.
4. Donnan Equilibrium Experiment:
Take two beakers of 100 ml capacity and label them as A and B. Into beaker A, introduce 50 ml
of 2% gelatin solution and in beaker B, put 50 ml of distilled water. Add 1 ml of thymol blue
indicator solution in each of the beakers. Now in beaker B, add 0.1N HCl drop by drop until the
colour of the solution becomes light pink (shade between yellow and pink). Note the pH of this
solution which should be around 2.0. Similarly add 0.1 HCl to beaker A so that the colour of the
gelatin solution also assumes light pink shade. Try to match the colours of the solution in both
the beakers. The pH of both solutions would also be same. Now transfer gelatin solution of
beaker A to a dialysing bag and suspend it in beaker B, containing distilled water. Let the bag
stand for some time. After a short while the gelatin solution in the bag will gradually turn yellow
and the surrounding solution will turn deep pink. Yellow colour is due to increase in pH of the
solution (decreasing acidity) and deep pink colour is due to decrease in pH (increasing acidity).
This indicates that ions have diffused out of the bag.
5. Ringers solution was used to provide enough Na+ and K+ ions needed to initiate sodium
potassium pump. It also has the same osmotic pressure exhibited by animals and thus
comparable to amphibian tissues physiological condition.
6. Sodium potassium pump active transport system sets and maintains the intracellular
concentrations of Na+ and K+ and for generating the transmembrane electrical potential. For

every 3 Na+ out, there will be 2 K+ that will move in. And for each molecule of ATP converted to
ADP and Pi, there will be 2 K+ ions that will move in and 3 Na+ ions out across the plasma
membrane.

a. 3 Na+ ions inside the cell will bind to the

Na+ - K+ ATPase enzyme
b. Conformation will change via
phosphorylation of the enzyme
stimulated by ATP
c. Expulsion of 3 Na+ outward as 2 K+ bind
from the outside of the cell
d. Stimulation of dephosphorylation of the
enzyme
e. Restoration of original confirmation of
the enzyme
f. Release of 2 K+ into the cell

The conversion of ATP to ADP and Pi is catalyzed by the enzyme, involving formation and hydrolysis
of the phosphoenzyme:
(1) ATP + Enz I ADP + P-Enz II
(2) P-Enz II + H2O Enz I + Pi
______________________
ATP + H20 ADP + Pi
7. Possible mechanism:
The barrier epithelia of amphibians have tight junctions of high electrical resistance between the
outermost living cells which render the epithelium "tight" to paracellular flow of Na+ or K+, thus
allowing vectorial transepithelial transport of these ions. The Na+absorption function is carried
out by granular or "principal" cells via Na+ channels in the apical membranes and an Na,KATPase pump in the basolateral membranes; K+ is recyled across this barrier via K+-selective
channels. The multilayered epithelium of frog skin functions as a syncytial Na+ transport
compartment. The granular cells, along with the deeper spinosum and germinativum cells, form
the syncytium. The cells in deeper layers are coupled to the first reactive cell layer by gap
junctions and although the cells in the different layers share the Na transport load, only the
granular cell layer has a polarized apical membrane containing amiloride-sensitive Na+ channels.
The amphibian skin and urinary bladder actively secrete hydrogen ions under appropriate
electrochemical gradients. Active H+ secretion is restricted to the mitochondria-rich cells.
8. If the skin bag is turned proper side out, the side of skin exposed to the medium reversed the
direction of the movement of ions. Thus, sodium ions will move out f and potassium ions will
move in. Alteration of results will be due to the composition of the skin layer. A decrease in the

weight of the bag will also be observed because the molecular weight of K+ ions is lower than
that of Na+ ions.
9. Cyanide ions are inhibitors of electron transport system since it binds in the ion in the the
cytochrome c oxidase. As a result, it blocks the flow of electrons from NADH-H+/FADH to oxygen
and stops ATP generation. in the absence of ATP, sodium potassium pump also stop and thus
inducing no change in weight of the skin bag.
10. The average rate of active transport for the set up without an inhibitor is negative while that
with inhibitor (NaCN) is positive. The weight of the skin bag exposed to Ringers solution alone
decreases while the weight of the skin bag exposed with Ringers solution and cyanide increases.
Theoretically, the weight of the skin bag exposed to NaCN should not change and reach
equilibrium. Due to some human errors like measuring that depends on the visual selection of
the student and maybe leaks on the skin bag, the results differs to the theoretical one.