Yellapragada Subbarow: The Chemist Who Found ATP and Phosphocreatine

Every second, every cell in your body uses adenosine triphosphate, ATP, as its energy currency. The molecule was found in 1929 in muscle extracts by two research groups: Karl Lohmann in Germany, and, independently, Cyrus Fiske and Yellapragada Subbarow (1895-1948) at Harvard Medical School in Boston, USA. Subbarow, an Indian biochemist from Andhra Pradesh, also discovered phosphocreatine (creatine phosphate) with Fiske, and developed the Fiske-Subbarow method, a colourimetric test for phosphate that became a worldwide standard in laboratories for decades. Later, at Lederle Laboratories, he led the team that synthesised folic acid and developed methotrexate, one of the first effective anticancer drugs, and supported the discovery of the antibiotic tetracycline (Aureomycin, chlortetracycline). His work is a landmark of Indian contribution to biochemistry, though he never received a Nobel Prize.

The Scientific Problem Before the Discovery

By the 1920s, scientists knew that muscle contraction produces lactic acid, and that phosphate compounds are involved. But how does muscle store and release energy? Embden and Meyerhof had studied glycolysis. Yet the immediate source of energy for contraction was unknown, and chemists had no simple, reliable way to measure small amounts of phosphate in blood, urine and tissue extracts. Without a good analytical method, the chemistry of phosphorus compounds in living tissue could not be studied properly.

The Discovery / Experiment

Step 1: A better phosphate test

In 1925, Fiske and Subbarow published a colourimetric method to determine inorganic phosphate. The principle is based on the formation of phosphomolybdic acid:

PO43- + 12 MoO42- + 24 H+ → [PMo12O40]3- + 12 H2O
(phosphomolybdate)  + reducing agent (aminonaphtholsulphonic acid) → molybdenum blue

In acidic ammonium molybdate, phosphate forms phosphomolybdic acid, which a mild reducing agent (Fiske and Subbarow used 1-amino-2-naphthol-4-sulphonic acid) reduces to an intense blue compound. The colour intensity is directly proportional to phosphate concentration (Beer-Lambert law), so it can be read in a colourimeter. The method was accurate to a few micrograms and was published in the Journal of Biological Chemistry.

Step 2: Phosphocreatine

Using the new method, Fiske and Subbarow analysed muscle extracts. In 1927 they found that muscle contains a labile phosphorus compound, which breaks down easily in weak acid, releasing inorganic phosphate in the process. They isolated it as a barium salt and identified it as phosphocreatine (also called creatine phosphate). They saw that its level falls when muscle contracts and rises again during recovery. They proposed that it was the immediate energy reservoir of muscle.

Phosphocreatine + ADP  ⇌  Creatine + ATP   (creatine kinase)

Step 3: Adenosine triphosphate

In 1929, they isolated from muscle a second acid-labile phosphorus compound containing adenine, a pentose and phosphate groups, and determined that it was an adenylpyrophosphate, i.e. ATP, with three phosphate groups, two of them easily hydrolysed. Karl Lohmann in Heidelberg (Meyerhof's laboratory) reported ATP at the same time, and so the discovery is credited to both groups. At first Fiske and Subbarow's paper described the compound as an "adenylpyrophosphate", and they suggested its role in muscle contraction.

ATP + H2O → ADP + Pi     ΔG°' ≈ −30.5 kJ/mol
ADP + H2O → AMP + Pi
ATP + H2O → AMP + PPi

Fritz Lipmann in 1941 later developed the concept of ATP as the universal energy currency ("high-energy phosphate bond"), and Szent-Györgyi showed that ATP makes actomyosin contract.

Step 4: Later work at Lederle Laboratories

Subbarow joined Lederle Laboratories (American Cyanamid) in 1940 as Director of Research. His team:

• Synthesised folic acid (pteroylglutamic acid) in 1945, which cures megaloblastic anaemia.
• Developed folic acid antagonists such as aminopterin and later methotrexate, which Sidney Farber used in 1948 to induce remission in children with acute leukaemia.
• Discovered diethylcarbamazine (Hetrazan) for the treatment of filariasis, a disease of great importance in India.
• Brought the antibiotic chlortetracycline (Aureomycin), discovered by Benjamin Duggar, at Lederle, where Subbarow led the research programme that developed this first tetracycline antibiotic (announced in 1948).

The Chemistry Behind It

• ATP structure: adenine + ribose (adenosine) + three phosphate groups joined by phosphoanhydride bonds.
• High-energy bond: hydrolysis releases about 30.5 kJ/mol under standard biochemical conditions because of relief of charge repulsion, resonance stabilisation of products and better solvation.
• Phosphocreatine: a guanidinium phosphate (N-phosphoryl) with a larger free energy of hydrolysis (about −43 kJ/mol) than ATP, so it can rephosphorylate ADP quickly.
• Folic acid: pterin ring + p-aminobenzoic acid + glutamate; its reduced form tetrahydrofolate carries one-carbon units for nucleotide synthesis.
• Methotrexate: a structural analogue that binds strongly to dihydrofolate reductase, blocking thymidylate and purine synthesis in fast-dividing cells.

Applications Today

• Clinical biochemistry: Molybdenum blue-type phosphate assays are still used in water analysis, soil testing and blood tests.
• Sports nutrition: Creatine supplements are used to raise phosphocreatine stores for short, intense exercise.
• Cancer therapy: Methotrexate is on the WHO list of essential medicines and is used for leukaemia, lymphoma and other cancers, and in lower doses for rheumatoid arthritis and psoriasis.
• Public health in India: Folic acid supplements in pregnancy prevent neural tube defects; diethylcarbamazine is used in the national filariasis elimination programme.
• Antibiotic therapy: Tetracycline-class drugs like doxycycline are standard treatments.

Exam Corner

• Fiske-Subbarow method: phosphate + ammonium molybdate gives phosphomolybdate; reduction gives molybdenum blue (measured colourimetrically).
• ATP = adenosine + 3 phosphates; ΔG°' of hydrolysis about −30.5 kJ/mol.
• Phosphocreatine + ADP gives creatine + ATP (creatine kinase) and acts as the immediate energy buffer in muscle.
• Methotrexate inhibits dihydrofolate reductase (DHFR).
• Folic acid has pteridine, PABA and glutamate; deficiency causes megaloblastic anaemia.

Common Misconceptions

• "Subbarow alone discovered ATP." ATP was isolated independently by Fiske and Subbarow and by Lohmann in 1929.
• "ATP is stored in large amounts." The body stores only a few seconds' worth; it is constantly regenerated.
• "Subbarow won a Nobel Prize." He did not, though many scientists believe he deserved wider recognition.

FAQ

Who discovered ATP?

ATP was discovered in 1929 independently by Karl Lohmann and by Cyrus Fiske with Yellapragada Subbarow.

Why was the Fiske-Subbarow method important?

It gave the first simple, reliable way to measure small amounts of phosphate, making modern biochemistry of phosphorus compounds possible.

How is Subbarow connected to cancer treatment?

His group at Lederle developed folic acid antagonists, including aminopterin and methotrexate, which are used in chemotherapy.

Which exam topics come from his work?

ATP structure and energetics, phosphocreatine, colourimetric analysis, folic acid, and anticancer antimetabolites are all common in JAM, NET and GATE.

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