101
Discussion and Conclusion
It is normal to use molecular models to illustrate the important
structural features of complex molecules and simple demon-
strations can be devised to illustrate complex macromolecular
motions and operating forces to the immense benefit of students.
This is the case with the DNA unwinding process where we have
designed a simple experiment to demonstrate the forces and
motions involved in the gyration generated in front of the
replicating fork. The experiment shows that when an unwinding
force is applied to a double-stranded helix, gyration ensues first
in one direction and then in the opposite direction, finally
resulting in considerable entanglement of the string.
In trying to relate the above observation to actual DNA
unwinding, the roles played by the helicases and topoisomerases
have to be explained. The initial force applied to the string is
analogous to the unwinding force applied by helicase to the
double stranded DNA. This analogy is justifiable because
helicases act in a wedgelike m a n n e r ' a n d consume about two
ATP molecules per base-pair separated. 3"9 This is a considerable
amount of energy and is enough to induce torsional strain in
front of the replicating fork.
Topoisomerase I functions by nicking and resealing one strand
only whereas topoisomerase II acts on both strands. "~ The
introduction of nicks relieves the torsional strain and prevents
strand breakage. In the first instance then, it would be
reasonable to assume that the initial rotation would be prevented
by topoisomerase I and this action requires no energy. With the
torsional strain relieved, there would probably be no energy left
for the reverse rotation and therefore no need for the action of
topoisomerase II. It appears then, in this case, that topoiso-
merase II is required mainly for the refolding of DNA.
In summary, this simple demonstration, using unwaxed twine
or nylon fishing lines, illustrates the unwinding process in front
of a replicating fork. It should be emphasized that this is what
ought to happen, but in reality, within the nuclei, the DNA is
very long, never relaxed along its whole length and capable of
breaking if allowed to gyrate. Topoisomerases are therefore
used to relieve the torsional strains and consequently prevent the
gyration observed in the experiment. In fact, the first topoiso-
merase (type II) discovered was of bacterial origin and was
named gyrase II to reflect its function. The demonstration
therefore indirectly illustrates the functions of the topo-
isomerases.
References
~Watson, J D and Crick, F H C (1953) Nature 171,737-738
-"Banfalvi, G (1986) Biochem Educ 14, 50-59
~Stryer, L (1981) Biochemistry, Freeman, New York
4Murray, R K, Granner, D K, Mayes, P A and Rodwell, V W (1990)
Harper's Biochemistry, Appleton and Lange, Norwalk, NJ
5Banfalvi, G (1984) Biochem Educ 12, 155-156
~'Alberts, B, Bray, D, Lewis, J, Raft, M, Roberts, K and Watson, J D
(1983) The Molecular Biology of the Cell, Garland, New York
7Banfalvi, G (1986) Biochem Educ 14, 7-10
8Fieldhouse. J (1981) Biochem Educ 9, 88
"Kornberg, A, Scolt, J F and Bertsch, L L (1978) J Biol Chem 253,
3298-3304
"~Cozzarelli, N R (1980) Seience 207. 935-960
IIGellert. M, Mizuuchi, K, O'Dea, M H and Nash, H A (1976) Proc
Natl Acad Sci USA 73, 3872-3876
BIOCHEMICAL EDUCATION 22(2) 1994
0307-4412(94) E0027-L
Changes in Carbohydrate Content During Fruit
Ripening m A New Approach of Teaching of Carbo-
hydrate Chemistry in Biochemistry Course
PORNTIP CHAIMANEE and ORANART
SUNTORNWAT
Department o f Chemistry
Faculty o f Science
Silpakorn University
Nakorn Pathom 73000
Thailand
Introduction
Fruit ripening is characterized by a series of coordinated changes
in the biochemistry and physiology of the tissue involved. ~ Fruit
flavor is based mainly on the balance between sugar, organic
acids and numerous other aromatic compounds. The hydrolysis
of starch during ripening to produce sugars was reported in
various fruits.- " Decrease in acidity concomitant with increase
in sugars is also observed during ripening. 4 The sweetness of
fully ripe fruit is due to the high level of sugars.
The experiment described here was designed for senior
chemistry students to enable them to handle their own exper-
iment and to encourage them to think about the possible
correlation between the biochemical processes, especially in
carbohydrate metabolism, that lead to change in fruit flavor
Students should learn the strategies in carbohydrate analysis
during the period of fruit ripening. The choice of fruits is
justified as follows: (i) they have a high starch content that may
easily demonstrate the drastic change during ripening, (ii) they
are cheap and easy to obtain.
Experimental
Plant material Fresh mature firm mangoes were obtained from
local market and were ripened at 24C. During ripening, fruit
pulp was collected everyday and frozen at -20C until use. If
fresh samples are readily available during the time of the
experiment, the pulp from different stages of fruits could be
collected right away without freezing
Alcohol insoluble solid (AIS) Fresh or frozen fruit pulp (20 g)
is homogenized in 100 ml hot 95% ethanol for 15 min. The
homogenate is allowed to cool down, is filtered and the residue
washed twice with ethanol followed by acetone. The samples are
then dried to constant weight under vacuum. The dry samples
are ground in a coffee grinder and kept frozen until used.
Fruit juice Fruit pulp from fresh or frozen samples (10 g) is
homogenized in 50 ml distilled water and clarified by centri-
fugation at 15 000 x g, 20 min. The supernatants are stored
frozen until used.
Starch determination The starch content is determined by the
method of Schmieder. 5 AIS (l g) is dissolved in 70 ml calcium
chloride solution (600 g CaCI2.2H20 in 700 ml distilled water),
boiled for 30 rain and the volume adjusted to 100 ml with
calcium chloride solution. The appropriate dilution of AIS
solution was made and then reacted with 0.1 mi of KI-Iz
solution in a final volume of l0 ml. The absorbance at 590 nm of
the AIS sample is recorded together with those of standard
starch solutions.
Total soluble sugar and reducing sugar determhtation Total
soluble sugar of fruit juice is determined by the phenol-sulfuric
method and reducing sugar by the Somogyi-Nelson method
102

Sugars The sugars in fruit juice are analysed and identified Table 1 Composition changes during ripening of Nam-Dokmat
using thin layer chromatography on silica gel with n-propanol/ mallgoes
acetone/l M lactic acid (7:1:1) as the mobile phase. The
chromatogram is then sprayed with aniline-diphenylamine- Total Reducing Titratahle
phosphoric acid reagent. Quantitative analysis of each sugar spot Dto'(s) after Starch soluble sugar acidity
is also performed by densitometry. harvest (mg/g AIS) (mg/g FW) (mg/g I~W) (mEq 101)g i ) pH

1 221).3 100 35.8 6,0 3.4

p H and titratable acid The pH of the fruit juice is determined 2 259.1) 118 38.1) 7.0 3.4
by pH meter. Titratable acid is determined by titration with 3 118.1) 155 38.3 6.7 3.4
0.1 M NaOH and expressed as mEq 100 g-i fresh weight. 4 33.3 155 39.2 3.8 3.7
5 4.0 161 43.6 2.0 4.3
Timetable 6 1.8 197 43.8 1.7 4.5
This experiment can be completed in two periods (4-h period) 7 1.2 2112 43.5 0.7 4.7
and the students can work together in groups of 2 to 3.
100
Session 1 The students will be able to prepare all the reagents
needed and then go on to the preparation of fruit juice and AIS. Sucrose /
80 . Fructose
Session 2 The students will determine the starch content from
AIS, the amount of sugar and reducing sugar from fruit juice and 60
also TLC.
"~ 40
A discussion session on the theoretical background to fruit
ripening should be arranged for the students. At the same time
they should be able to compare their results in graphic or tabular 20
form and speculate on the biochemical mechanisms behind the ~ -

sweetness in ripe fruit. I I L I I I

Results
During the ripening process, sugar content increased while
acidity decreases (Table 1). The amount of sugar doubles after 7
days of storage while the amount of titratable acid is significantly
decreased by day 7. These trends are the consequence of the
change in sugar:acid ratio, which increases throughout the
ripening process. Starch depletion and increment in the amount
2 3 4 5 6 7
Storage day
Figure 2 Changes in sugar content o f mangoes during ripening
of reducing sugar during ripening is also observed. Typical
chromatographic separations are shown in Figure 1. The change
in sugar content during ripening are shown in Figure 2. Sucrose
predominated throughout the 7 day periods, with fructose as the
major reducing sugar.

Discussion
Students have shown a great interest in this experiment which
OeJ
enables them to develop qualitative and quantitative insights
into the correlation of starch depletion and increase in sugar
content with decrease in organic acids in fruit which lead to
sweetness during the ripening process. Fruit models can also be
explored in other aspects of carbohydrate chemistry such as
changes in cell wall component related to fruit texture during
o ripening. Furthermore, the experiment can be performed using
other locally or seasonally-available fruits such as banana, peach
and tomatoes.

Acknowledgements
The authors thank the IDP program for its support to PC and OS study
visit (19911, 1993) and collaborative research fund 11993)

! /
References
I Brady, C J (1987) Ann Rev in Plant Physiol 38, 155-178
"Pech, J C and Latchke, A 11972) J Sci Food Agric 23, 1499-15112
Fuch, Y, Pesis, E and Zanberman, G (1980) Scientia Hortic 13, 155-161)
~Morga, N S, Lustre, A O, Tunac, M M, Bologot, A H and Soriano, M
R (1970) Food Chem 4, 225-235
>Schmieder, R L and Keeny. P G (19811)J Food Sci 36, 486-489

Figure I TLC separation (scan) o f sugars from mango fruit

B I O C H E M I C A L EDUCATION 22(2) 1994