Studies on the conversion of indole acetic acid and oxindole acetic acid by Hygrophorous conicus

94 Pages·2017·1.99 MB·English

by  Barbara Ruth Patterson

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Masters Theses Student Theses and Dissertations
1965
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Barbara Ruth Patterson
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Patterson, Barbara Ruth, "Studies on the conversion of indole acetic acid and oxindole acetic acid by
Hygrophorous conicus" (1965). Masters Theses. 5336.
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STUDIES ON THE CONVERSION OF
INDOLE ACETIC ACID TO OXINDOLE ACETIC ACID
BY HYGROPHOROUS CONICUS
BY
BARBARA R. PATTERSON
A
THESIS
submitted to the faculty of the
UNIVERSITY OF MISSOURI AT ROLLA
in partial fulfillment of the requirements for the
Degree of
MASTER OF SCIENCE, CHEMISTRY MAJOR
Rolla, Missouri
1965
Approved by
ii
Abstract
The fungus. Hygrophorous conicus, produces the enzyme,
indole acetic acid oxidase, when grown in the presence of indole
acetic acid (IAA) or tryptamine. The IAA acts both as an inducer
and a substrate for this intracellular enzyme. The product of the
oxidation catalyzed by the IAA oxidase is oxindole acetic acid (OIAA).
Since the IAA and OIAA absorb maximally at two different
wavelengths the use of spectrophotometric analysis was possible for
assaying for the IAA oxidase activity. Paper chromatography was
used for qualitative identification of the IAA and OIAA.
The rate of growth and the rate of formation of the enzyme by
H. conicus were studied. The rate of growth was found to be similar to
that of other fungi. The formation of the enzyn1e, however, proved
to be somewhat different from that of other inducible enzyme systems.
The OIAA formed by the oxidation of IAA was also found to in
duce the formation of an enzyme in H. conicus. This enzyme catalyzed
the destruction of OIAA.
Several methods were tried in an attempt to isolate the lAA
oxidase from the cells of H. conicus, but these methods proved, for
the most part, unsuccessful
iii
TABLE OF CONTENTS
Page
TITLE PAGE. ...... . i
ABSTRACT ........ . ii
TABLE OF CONTENTS. iii
LIST OF FIGURES . v
LIST OF TABLES vi
I. INTRODUCTION . 1
II. LITERATURE REVIEW. 2
Biological Oxidation ...... . 2
IAA Oxidases and Peroxidases 3
Nature of the Enzyme ..... . 5
Preparation of a Cell-Free Enzyme. 6
Enzyme Assay ........... . 8
Spectrophotometric Analysis 9
Colorimetric Analysis . 12
Manometric Methods .. 12
Paper Chromatography. 13
Enzyme Induction ...... . 13
III. EXPERIMENTAL. 16
Materials ... . 16
Apparatus ... . 17
General Methods 19
Preparation of the Sodium Salt of Indole
Acetic Acid ................. . 19
Cultivation of H. Conicus. ........ . 19
Preparation of Two Dimensional Paper
Chromatograms .... . 22
Enzyme Assay ....... . 24
Method 1: Extraction 34
Method 2: Filtration .. 35
The Rate of Growth of H. Conicus and
Formation of the Enzyme ... . 36
OIAA Induced Enzyme ........... . 40
46
Effect of pH on Enzyme Activity .... .
Methods for the Preparation of Cell-Free
50
Enzyme ...................... .
Biuret Test for Protein .......... . 52
Spectrophotometric Method for Protein
Determination ....... . 53
Freeze Drying of the Cells. 53
Insonation of the Cells .... 59
lV
Page
IV. DISCUSSION . . . . ..................... . 65
Preparation of the Sodium Salt of Indole Acetic
Acid ......... · · · · · · · · · · · · . . . . . . . 65
Enzyme Assay. ...... · · . . . . . . . . . . . . . . 65
Rate of Growth of H. Conicus and Formation of
the Enzyme. . . . . . . . . . . . . . 66
OIAA Induced Enzyme. . . . . . . . . 69
Effect of pH on Enzyme Activity. . 70
Preparation of Cell-Free Enzyme. 71
Spectrophotometric Method for Protein
Determination. . . . . . . . 71
Freeze Drying of the Cells 72
Insonation of the Cells 73
V. CONCLU@ONS . . . . . 74
VI.' RECOMMENDATIONS 75
VII. BIBLIOGRAPHY . . . . 76
VIII. ACKNOWLEDGEMENTS . 79
IX. VITA ......• 80
APPENDIX 1 81
APPENDIX 2 85
v
LIST OF FIGURES
Figure Page
1 Typical Chromatogram of IAA, OIAA and
5-Hydroxy IAA .............. . 25
2 Absorbance of IAA and OIAA in Water
Determined on the Beckman DK-2A . 27
3 Absorbance of Various Concentrations of IAA
at 280 and 250 mfl. ................. . 30
4 Absorbance of Various Concentrations of OIAA
at 25 0 and 280 m fl. ................ . 31
5 Absorbance of Various Mixtures of lAA and
OIAA ...................... · . 33
6 Plot of the Dry Weight of Mycelia versus
Sampling Time . . . . . . . . . . . . . . . . . 39
7 Rate of Formation of IAA Oxidase. 43
8 Standard Curve for Biuret Determination. 55
9 Nucleic Acid Concentration and F Values at
Various 280/260 Ratios ............ . 57
vi
LIST OF TABLES
Table Page
l Comparison of IAA and NaiAA as Inducers ... 21
2 Absorbance Values of IAA and OIAA in Water
at Various Wavelengths Determined Using
the Beckman DK-2A Spectrophotometer. · · · . 26
3 Absorbance Values for Various Concentrations
of IAA and OIAA in Water ............. . 29
4 Absorbance Values for Mixtures of IAA and OIAA .. 32
5 Dry Weight of Mycelia at Various Sampling
Times ••....................... 38
6 Enzyme Activity at Various Times for Run l 41
7 Enzyme Activity at Various Times for Run 2 . 42
8 Spots Found on Paper Chromatograms Prepared
from the Spent Malt Mediwns and Assay
Samples ........................... . 44
9 Contents of Flasks Used for As say in OIAA
Induced Enzyme Experiment ..... . 45
10 Enzyme Activity and Results of Paper
Chromatograms for the OIAA Induced Enzyme
Experiment ....................... . 47
ll Preparation of Buffers. 48
llA Preparation of Various pH Buffers. . . . . . . . . . . . 49
12 Effect of pH on Enzyme Activity .. 51
13 Biuret Determination of Protein Standard 54
14 Nucleic Acid Concentrations and F Values at
Various 280/260 Ratios ........... . 56
15 Enzyme Activity of Freeze-Dried Cells for
Run l ....................... . 60
vii
Table Page
15A Enzyme Activity of Freeze-Dried Cells for
Run 2 ........................ . 61
16 Enzyme Activity of Insonated Cells 63
APPENDIX
1-A Absorptivities of IAA and OIAA 84
2-A The Absorbance Values at 250 and 280m~ for
IAA Standards .................... . 86
1
I. INTRODUCTION ~
. ( OJ"
I
CHz-COOH .
Indole acetic ac1d h. 1s a growthhormone
N
H
which is widespread in nature. Indole acetic acid oxidase, an enzyme
which catalyzes the oxidation of indole acetic acid, is almost as wide-
spread in nature as indole acetic acid. Indole acetic acid oxidase has
been found in many plants and in several fungi. One of the fungi in
which this enzyme has been found is Hygrophorous conicus. The indole
acetic acid oxidase of H. conicus is an induced,intracellular enzyme.
enzyme is of interest because of the unique oxidation product and also
because it is one of the few indole acetic acid oxidase systems for
which the oxidation product has been isolated and positively identified.
The purpose of this research project was to develop a more
satisfactory method for assaying the indole acetic acid oxidase activity
in H. conic us, and to develop a method for preparing a cell-free ex-
tract of the enzyme. Although the latter objective was not fullyattained,
several interesting facts were discovered concerning the fungus, H.
conicus, and the enzyme activity.
2
II. LITERATURE REVIEW
Biological Oxidation
Biological oxidation proceeds by many different mechanisms
and is catalyzed by several different groups of enzymes. Some of
the groups which catalyze oxidations are oxidases, peroxidases,
oxygenases, and dehydrogenases.
Oxidases catalyze reactions in which oxygen molecules serve
as the immediate electron acceptor (Hayaishi, 1962). Classical
oxidases may be divided into two categories. In the first category
the enzyme catalyzes the transfer of two electrons from the substrate,
S, to one molecule of oxygen-forming hydrogen peroxide as sho.vn in
equation ( 1).
( 1)
In the second category two electrons are transferred to one
atom of oxygen to produce water as seen in equation (2 ).
SH2 + 1/2 o2 oxidase)- S + H20 (2)
o
Peroxidases are enzymes which use H as an acceptor of
2 2
electrons as shown in equation {3 ).
+ +
peroxidase) S {3)
In oxidation reactions in which oxygen is directly incorporated
into the substrate molecule as in equations (4) and (5 ), the enzyme
catalyzing the reaction is called an oxygenase.