Robert Joachim Feulgen
Birth Date: September 2, 1884
Death Date: October 24, 1955
Place of Birth: Werden, Germany
Nationality: German
Occupations: chemist
Although German physician
Robert J. Feulgen published seventy research papers about the chemistry
of physiology, histochemistry, and wrote a review of nucleic acid
chemistry titled Chemie und Physiologie der Nucleinstoffe (Chemistry
and Physiology of Nucleic Acids), he is best known for his discovery of
a method for staining nucleic acid, now termeda Feulgen reaction.
Feulgen was born in Werden,
Germany, into a working class family, Feulgen paved his way to the
medical school through an outstanding student performance that opened
to him the doors of the University of Freiburg at Breisgau in 1905. His
medical residency was taken in the City Hospital of Kiel, where he
wrote his dissertation on the purine metabolism of patients with
chronic gout. After finishing residence, he moved to Berlin and started
his carrier as an experimental researcher at the Chemistry Department
of the Physiological Institute, then under the direction of Hermann
Steudel, a nucleic acid chemist.
Feulgen worked with
Steudel from 1912 until 1918, improving Steudel's technique for
thymonucleic acid staining via biuret reaction. At the time, what later
became known as DNA, was termed thymonucleic acid and thyme tissues
were used for the extraction of nucleic acids. Biuret, a compound
obtained from urea, gives proteins a blue-violet color (biuret
reaction), when copper sulfate and strong alkali are added without
heating, and it was used by Steudel to stain DNA. However, the color
was lost when water was added, resulting in a colorless solution.
Feulgen introduced a new staining technique by adding Congo red
combined with malachite green and extracting the salts formed, thus
avoiding the biuret reaction and the loss of staining. This new
staining technique allowed a more reliable analysis of the resulting
product and the calculation of nitrogen and phosphorus ratios present
in nucleic acids.
Based upon his
observations, Feulgen defended the hypothesis that nucleic acids were
formed by four nucleotides, which at the time was thought by many to be
too few units to carry the enormous wealth of information needed to
transmit genetic information.
In 1914, Feulgen made a major
contribution to the tetra nucleotide hypothesis (i.e., four nucleotides
forming an oligonucleotide) when he discovered that phenyl hydrazine
reacted with apurinic acid, what indicated the presence of aldehyde
groups.
Using aldehyde-blocking
controls, Feulgen found that when thymonucleic acid (now known as DNA)
is mildly hydrolyzed to apurinic acid, the loss of purines exposes
aldehyde groups. He termed his discovery the nucleal reaction, since it
is a reaction specific for aldehydes based on the formation of a
magenta-colored compound, when aldehydes react with fuchsin-sulfuric
acid. The positive reaction occurs only in the presence of
deoxyribonucleic acid (DNA) whereas yeast nucleic acid (RNA) did not
give the same reaction.
In 1919, Feulgen defended his
doctorate thesis on the state of the art of nucleic acid research,
where he also made a critique of Steudel's staining technique. In the
same year, after receiving his Doctor of Philosophy (PhD) degree, he
was offered a research position in the Physiological Institute at
Giessen, where his career gradually unfolded through successive
promotions, until he was named director of the Physiological-Chemical
Institute in 1931.
Feulgen further applied the
nucleal reaction as a histochemical staining assay that confirmed that
such reaction only takes place in the cell nuclei of both plants and
animals. He was not able to obtain a positive nucleal reaction in
pentose nucleic acid obtained from yeast cells, but he rightly inferred
from several studies that the pentose nucleic acid (RNA) of yeast was
localized in the cytoplasm. Feulgen exposed during the Congress of
Physiology in Tübingen that year those results but his conclusions
were met with general skepticism.
In the 1920s there was a common
assumption that nucleic acids occurred under the form of thymonucleic
acid (DNA) only in animals and as pentose nucleic acid (RNA) only in
yeast and plants. Another study by Feulgen, in 1937, provided data
contradicting such hypothesis. He isolated rye germ nuclei and
succeeded in obtaining positive nucleal reactions, therefore proving
that DNA is present in the cells of both plants and animals. He also
inferred from these experimental studies with nucleal reaction that
thymonucleic acid (DNA) in Eukaryotes was located at the nucleus
whereas pentose or yeast nucleic acid (RNA) was located at cytoplasm.
Although we now know that RNA may also be present in the nucleus, being
synthesized there and then transported to the cytoplasm, Feulgen's
conclusion was insightful and substantially correct right regarding the
general localization of each type of nucleic acid in the cell.
In 1924, Feulgen and Voit
discovered a positive nucleal reaction in the cytoplasm without first
performing mild hydrolysis. They concluded that such outcome was due to
the presence of aldehyde groups in the cytoplasm. Since a further test
with the addition of lipid solvents was negative to nucleal reaction,
they inferred that a lipid precursor was responsible by the first
results obtained, and termed it plasmalogen. Four years later, they
isolated the plasmalogen and in 1939, they were able to identify it as
an acetal phosphatide.
Another important Feulgen
contribution to the understanding of the chemical e molecular structure
of DNA came in 1936 through his studies of the nucleic acids in their
undegraded state, which led him to conclude that nucleic acids were in
fact molecular polymers formed by
oligonucleotides, each
comprised of the bases thymine, cytosine, adenine and guanine.
Therefore, he also concluded that the extractive techniques utilized so
far resulted in a mixture of depolymerized fragments of such a
molecule. He studied the viscosity and the optical activity of the
polymer and established then two experimental states of the DNA
molecule, which he termed form A (undegraded molecule or polymer) and
form B (depolymerized molecule). He also succeeded in converting form A
to B by using a commercial pancreatic juice to digest the polymer into
fragments. The enzyme present in the pancreatic juice, responsible by
the depolymerization, was termed nucleogelase by Feulgen; and from the
same
commercial juice DNA
depolymerase was isolated some years later.
Although modern molecular
geneticists now appreciate the great importance of Feulgen's pioneering
studies, at his own time little attention was given to his experimental
work. The Feulgen reaction is still in use as an effective staining
technique in studies of chromosomes and in genetic cytochemistry
assays.
Further reading:
Mikroskopisch-chemischer
Nachweis einer Nucleisäure vom Typus der Thymonucleinsäure
und die darauf beruhende elektive Färbung von Zellkernen in
mikroskopischen Präparaten. R. Feulgen und H. Rossenbeck, Hoppe
Seyler Z Physiol Chem, 1924, 135, 203-248
The chemistry of Schiff's
reagent. F.H. Kasten, Int Rev Cytol, 1960, 10,
1-100
Mechanisms of the Feulgen acid
hydrolysis. P. Kjellstrand, J. Microsc., 1980, 119, 391-396
From Pixels to Picograms: A
Beginners' Guide to Genome Quantification by Feulgen Image Analysis
Densitometry . D.C. Hardie, T.R. Gregory, and P.D.N. Heberta, Journal
of Histochemistry and Cytochemistry, 2002, 50,
735-750
Robert Feulgen and his
histochemical reaction for DNA. F.H. Kasten,Biotech Histochem., 2003,
78, 45-49