Table Of ContentI S ^ 4
PURDUE UNIVERSITY
THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION
BY IRVING RICHARD SHERMAN.
ENTITLED STITDTRS DM (TERTATN PHAfiKS OF THF, KREBS' HYP.T.K____
______________IN LIUINfl-TTSSUF._______________________________________
COMPLIES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES
AND IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
Q - - ( ? Prt— P , Professor m Charge qf Thesis
Î
Hear of School or Department
TO THE LIBRARIAN:-----
-IS- •
THIS THESIS IS NOT TO BE REGARDED AS CONFIDENTIAL.
GRAU. SCHOOL. FORM 6
STUDIES ON CERTAIN PHASES OF THE KREBS' CYCLE
IN LIVING TISSUE
A Thesis
Submitted to the Faculty
of
Purdue U niversity
by
Irving Richard Sherman
In P artial Fulfillm ent of the
Requirements fo r the Degree
of
Doctor of Philosophy
January, 1951
ProQuest Number: 27714195
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ACKNOWLEDGMENTS
The author wishes to express his sincere thanks and ap
preciation to Dr, Ralph C, Corley who has always offered encouragement
and inspiration toward the successful prosecution of th is work. He
also wishes to thank his colleagues, Messrs. Fishbein, Graham, M ilionis,
R essler, Rimmer, Rosene and Taub without whose loan of equipment, space,
and tim e, the syntheses of many compounds would have been greatly pro
tracted. An expression of thanks is also extended to Mr. Harold A. Gamer
for his g ift of cis- and trans-aconitic acids. F inally, he wishes to
cite the cordial cooperation of his w ife, C harlotte, who put up with five
years of graduate school in right good humor and kept her husband in same.
TABLE OF CONTENTS
Page
ABSTRACT ............................................................................................................................... i
INTRODUCTION................................................................................................ 1
METHODS .................................................................................................................................. 3
EXPERIMENTAL...................................................................................................................... 20
DISCUSSION.......................................................................................................................... 51
SUMMARY ................................................................................................................................. 57
BIBLIOGRAPHY...................................................................................................................... 58
LIST OF TABLES AND FIGURES
List of Tables Page
1. Recovery Data on A Ipha-ketoglutarate afte r
Treatment with Ammoniacal S ilver N itrate ..................... 7
2. QO2 of Various Concentrations of C itrate in
the Presence of Calcium Ion ................ 21
3. Range of QO2 Values, Calcium Present, 120 Minutes ................21
4. Effect of Magnesium Ion on QO2, Calcium Absent .....................23
5. Range of QO2 Values, Calcium Absent with Various
Concentrations of Magnesium, 120 Minutes ....................................25
6. Typical QO2 Experiment, Run No. 37 .............................26
7. Effect of Inhibitors on QQg, Substrate Absent ..................27
8. Effect of Inhibitors on Q02,s, C itrate as Substrate .... 27
9. Effect of Inhibitors on QO2* s. Iso citrate as
Substrate ................................................................................................ 29
10. Effect of Inhibitors on Q02fs, C is-aconitate
as Substrate ................................................................................................. 31
11. Range of QO2 Values, Substrates with Inhibitors,
120 Minutes ........................................................ 31
12. Typical R.Q. Experiment, Run No. 44 .......................34
13. Effect of Inhibitors on QCO2'8 and R.Q.’s,
Tim% 120 Minutes ........................................................................ 35
14. Range of QO2 and QCO2 Values in R.Q. Experiments,
Time, 120 Minutes .................... 36
15. Alpha -ket oglut arat e Formed in the Presence of
Inhibitors ................................................................................................ 37
Figures
Transmittance of A Ipha-ketoglutarate
2,4-dinitrophenylhydrazone .................
QO2 of Various Concentrations of C itrate,
Calcium Present ..................................................................
Effect of Magnesium Ion on QO2, Calcium Absent 24
QO2 C itrate plus Inhibitors ......................................... 28
QO2 Iso citrate plus Inhibitors .......... . . . . . . 30
QO2 C is-aconitate plus In h ib ito rs ................. 32
R.Q., Basic Medium plus Inhibitors ......................... 38
R.Q. C itrate plus Inhibitors ............................................... 39
R.Q. Iso citrate plus Inhibitors ................................................ 40
R.Q. C is-aconitate plus Inhibitors ...................................... 41
R.Q. Substrates, No Inhibitors ................................................... 42
R.Q. Substrates plus A rsenite ....................................... 43
R.Q. Substrates plus lo d o acetate.............................................. 44
R.Q. Substrates plus Trans-aconitate ..................................... 45
R.Q. Substrates plus lodoacetate and Trans-aconita te .. 46
R.Q. Substrates plus A rsenite and lodoacetate .............. 47
R.Q • Substrates plus Arsenite and Tnans—aconitate .... 48
R.Q. Substrate plus A rsenite, lodoacetate and
Trans-aconitat e 49
STUDIES ON CERTAIN PHASES OF THE KREBS* CYCLE IN LIVING TISSUE*
By Irving Richard Sherman and Ralph C. Corley
(From, the Department of Chemistry, Purdue U niversity
Lafayette, Indiana)
AN ABSTRACT
INTRODUCTION
Work done in the la st decade (1,5 ,6,7 ,8,9>10) has fixed at
least the scaffolding structure of the c itric acid cycle. The question
now arises about possible alternate pathways of lesser metabolic im
portance . This work is an investigation into th at p o ssib ility dealing
specifically with those reactions leading from c itra te to alpha-keto-
glutarate.
METHODS
Rat liv er slices were used with the W arburg-Barcroft res-
pirom eter. The medium, 2.0 m l., either by its e lf or with substrates
and/or inhibitors dissolved in i t , was put into the center w ell of
a Warburg flask. The outer w ell contained 1.0 ml. 33% KOH solution
fo r most experiments. When keto acids were determined, 0.5 ml. KOH
solution was put into the outer w ell and 0.5 ml. 100% w/v trich lo ro
acetic acid was put into the side arm emptying into the center w ell.
When CO2 was being measured, water was substituted fo r KOH solution.
Readings for a ll the inhibitor studies were taken at 30
* Abstracted from the thesis of Irving Richard Sherman for the Doctor
of Philosophy degree.
minute intervals for 120 minutes. C ertain experiments carried out on
ionic composition were read only to 90 minutes and s t i l l other at 60
and 120 minutes only.
When keto acids were determined, the trich lo roacetic acid
was tipped in at the conclusion of the experiment. An additional 0.5
ml. water was added to bring the medium volume up to 3.0 ml. Analysis
of the keto acids was based on the method of Friedemann and Haugen (12).
It was modified by adding T ollen's reagent to destroy the pyruvate and
by diluting the colored solution to 12.5 ml.
The determ ination of alpha-ket oglut a rat e-ars enit e present as
an in h ib ito r is as follow s. To a 1.0 ml. aliquot in a centrifuge tube
add 1.0 ml. Tollen's reagent and heat in a water bath for ten minutes
at 40°. Centrifuge and decant into a te st tube. Add 3.0 ml. 10% t r i
chloroacetic acid solution to the centrifuge tube, mix, centrifuge and
decant into the te st tube. Treat the solution in a te st tube with 1.5
ml. 100 mg%. 2 ,4-dinitrophenylhydrazine in 2 N HC1 solution. Mix and
le t stand 25 m inutes. Add 5.0 ml. 2.5 N NaOH and le t stand 10 minutes.
Read on the Beckmann spectrophotometer at 400 mji setting a blank con
taining a ll the reagents at 100% transm ission.
We were interested in three systems:
(a) C itrate cis-aconitate ^ iso citrate
(b) Iso c itra te o x a lo su c c in a te
(c) O x alo su ccin atealp h a-k eto g lu tarate.
In (a) we inhibited aconitase by the use of 0.03 M trans-
ac onit ate. Trans-aconitate, 0,06 M, has been reported by Saffran and
Prado (13) to completely in h ib it aconitase in homogenates. This con
centration represents an osmolarity of 0.30 which is too high for whole
