Dr SubbaRow enrolled himself at the Harvard Medical
School Department of Biochemistry in the summer of 1924.
Professor Otto Folin, head of HMS Department of Biochemistry, was
then directing the talents of all in his department to development
of methods for analysing small quantities of blood, urine and tissue.
One such method developed in the department was for estimation of
phosphorus in tissues. It was however unsatisfactory in many ways,
and Folin assigned SubbaRow to assist Cyrus Hartwell Fiske, associate
professor, in removing the difficulties with the phosphorus method.
The task that Fiske gave SubbaRow was to search for a chemical
agent that would, in small concentrations, completely convert phosphorus
in the prepared tissue into a blue substance within the shortest
possible time. The agent should not be affected by other tissue
substances or by trichloro-acetic acid used for removing protein
from the tissue and should not require prior digestion of phosphorus
with sulphuric acid.
It was a challenge that held SubbaRow's interest right away. The
first satisfactory agent that SubbaRow found gave some 20 per cent
more colour than hydroquinone previously used in phosphorus estimation.
But it required 30 minutes to produce the maximum colour. Fiske
said the ideal agent would be one which did the job in just five
SubbaRow continued his search. An old sample of 1,2,6-amino-naphthol-sulphonic
acid gave very encouraging results. A purified sample of this turned
out to be 50 times more active than hydroquinone. It produced within
a fraction of a minute more intense colour than hydroquinone did
in 30 minutes. But it was a difficult chemical to prepare. Fortunately
it had an equally good isomer that could be prepared and purified
easily. This was 1,2,4-amino-naphthol-sulphonic acid which differed
only in the way the groups of atoms were arranged in the molecule.
It could also be readily bought in the market as it was in demand
from dyestuff makers who called it the 1,2,4-acid.
SubbaRow worked the next four months day and night to perfect the
phosphorus estimation procedure to the satisfaction of Fiske. The
1,2,4-acid stood every test. It did not require any big change in
the procedure for preparing the analytical material for colorimetric
estimation. It gave accurate readings even in the presence of ten
times more colour-inhibiting material than was permissible with
The “rapid colorimetric method for the estimation of inorganic
phosphorus, organic phosphorus, organic phosphates and lipoid phosphorus
in blood and urine”, SubbaRow recorded proudly, “is
correct to 1/100,000th of a grain”.
Fiske and SubbaRow were invited by the American Society of Biological
Chemists to demonstrate the new phosphorus method before its annual
meeting on December 29, 1924. Folin promptly asked SubbaRow to a
celebration dinner at his home and promised him a bigger fellowship
the following year.
The phosphorus procedure was presented to the Society as “Fiske-SubbaRow
Method of Estimation of Phosphorus”. SubbaRow had worked under
the supervision of Fiske. Besides, the association of a biochemist
of Fiske's reputation would help its ready acceptance by the profession.
SubbaRow was after all a novice, unknown to the scientific world
until then. The method is so accurate and suitable for analysis
of all kinds of biological and clinical material that it was adopted
immediately by biochemical laboratories all over the world. It is
used to this day in common analytical practice in many fields of
biochemistry. It is one of the first procedures biochemistry students