219
General Discussion
This experiment is intended to complement a lecture or seminar
on the chemical modification of enzymes. The recommended
size of class is 10-15 students. This experiment can be easily
adopted to single point determinations in a conventional
spectrophotometer when equipment availability for practical
classes in a limitation. The major advantages of this experiment
is that it is relatively easy and inexpensive and does not call for
special equipment. Currently our students work in groups of two
so that each student has something to do during the practical,
while they can share the observations and results within the
group.
Acknowledgement
One of the authors (JL) is thankful to Gulbarga University, Gulbarga
for providing facilities to conduct the experiment.
References
1Bernfeld P (1955) Methods Enzymol 1, 149-50
2Spande T F and Witkop B (1967) Methods Enzymol 2,498-532
3Dua R D and Kochhar Z (1986) Indian J Biochem Biophys 23,133-139
4Kochhar S and Dua R D (1985) Biochem and Biophys Res Commun
126, 966-973
5Pietkiewicz J, Kustrzeba W I, Wolna E and Wolny M (1987)
Biochemistry Internat 14, 805-814
0307-4412(95)00101-8
Creating Belousov-Zhabotinsky R e a c t i o n s in a Petri
Dish
M O N T S E R R A T F E R N A N D E Z - I N F A N T E S and
MIGUEL ANGEL MEDINA
Laboratorio de Bioquimica y Biologia Molecular
Facultad de Ciencias
Universidad de Mdtlaga
E-29071 M(daga, Spain
Introduction
The main indication of the presence of life is a profound
departure from equilibrium. I However, textbooks of Biochem-
istry and General Biology invariably obscure this fact by using
equilibrium or steady-state approaches when attempting to
explain the thermodynamics of biological processes. 2'3 Non-
equilibrium thermodynamics is generally absent from under-
graduate Biology courses. (If present at all it is usually restricted
to advanced Biophysics courses).
It is clear that a formal, rigorous presentation of, for example,
Prigogine's approach to non-equilibrium thermodynamics4 is far
beyond the scope of a course in general Biology. However, we
feel that the topic is so central to an understanding of biological
systems that some mention of it is desirable and that a
qualitative, intuitive approach is possible and is certainly better
than nothing.
A first step could be a study of the near-equilibrium reactions
which give rise to oscillatory behaviour. They demonstrate that
there is not always a simple asymptotic drive towards equi-
librium. Such reactions are important in Biology and photo-
synthetic and glycolytic oscillations are well-known,-5 but there
are simpler chemical examples known as 'chemical clocks'. This
paper describes a simple and cheap demonstration of an
oscillatory oxidation/reduction system.
Description of the Model System
The best known chemical clock is that provided by the
Belousov-Zhabotinsky (BZ) reactions. 6 These are autocatalytic
BIOCHEMICAL EDUCATION 23(4) 1995
oxidation/reduction reactions. There are many recipes for BZ
reactions but all involve reaction mixtures in which a periodic
change in oxidation state occurs and is maintained over a
number of cycles. These oscillations can be easily seen as
alternating changes in the colour of the reaction mixture of a
homogeneous preparation or as a travelling wave moving across
a two-dimensional preparation from the point where the reaction
was initiated.
Experimental
Students are provided with the following stock reagents: 0.25 M
KBrO3, 5 M H2SO4, 0.1% (w/v) methyl orange, 1 M NaBrO3,
1 M NaBr, 1 M malonic acid and 0.025 M ferroin. We suggest
the following two recipes for BZ reactions:
First recipe Prepare a reaction mixture with the following final
composition: 25 mM KBrO3, 50 mM H2SO4, 0.01% (w/v)
methyl orange. This mixture will be pink/red. Pour 20 ml into a
Petri dish. Add a single drop of 1 M NaBr to the center of the
dish. This changes the local pH giving a small paler pinkish
circle. This then grows and the rate of growth can be estimated
by placing millimetre graph paper under the dish. We have
found a more or less constant rate of about 6 mm/min. In the
place where the drop was added, the colour becomes paler and
paler and eventually becomes a yellowish dot. This then starts to
grow and spreads outwards in increasing circles. The colour at
the centre then becomes pinkish and the next wave spreads out
and the radiating pattern of alternating pinkish and yellowish
waves is established.
Second recipe Prepare a reaction mixture with the following
final composition: 0.33 M NaBrO3, 0.12 M malonic acid, 60 mM
NaBr, 0.37 M H2804, and 3 mM ferroin, When ferroin is added,
a dark precipitate is formed. Mix well until all of the precipitate
is dissolved giving a brown/orange solution. Pour 10 ml into a
Petri dish. A travelling wave is created by touching the solution
with a silver wire. Alternate brown/orange and blue/grey waves
are formed. When they reach the edge of the dish, they are
reflected back towards the centre (rather like the echo of a sound
wave).
Complex patterns can be created by touching the surface of
the mixture in different places and interference patterns occur
when different waves collide. Spiral patterns can also be set up. 7
Conclusion
The BZ chemical clock can be produced cheaply with a
minimum of apparatus and without lengthy laboratory prep-
aration. The oscillatory reactions and patterns of travelling
waves are eyecatching and arouse the interest of the students in a
way that perhaps mathematical expressions of non-equilibrium
conditions would not.
One of the coauthors (MFI) concerned with the optimisation
of the procedures described was a student on the General
Biophysics course at this University.
References
~Sagan C, Thompson R, Carlson R, Gurnett D and Hard C (1993)
Nature 365,715
2Rawn D J (1989) Biochemistry, second edition, Neil Patterson, New
York
3Lehninger A L, Nelson D L and Cox M M (1992) Principles of
Biochemistry, second edition, Worth, New York
4prigogine I (1980) From Being to Becoming, Freeman, New York
SVolkenshtein M V (1981) Biophysics, Mir, Moscow
6Hoppe W, Lohmann W, Markl H and Ziegler H (1982) Biophysics,
second edition, Springer, Berlin
7Walker Jearl (1978) Scientific American July 1978