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4. {ul I .IAJlID fill {III-I. ‘JI'lI {III III fl 1" I

EFFECT OF 2,h-DICHLOROPHEXOXYACETIC ACID ON
PHOTEOLYTIC ACTIVITY op RED KIaNaY BgAN PLANTS

(PHASEOLUS VULGARIs)

3?

Theodore Lynn Rebstock

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in purtlal fulfillment of the requirements
for the degree 0!

MASTER OF SCIENCE
Department of Chemistry

1951

EFFiCT 0F Z'Q-DI :hLOilCi r&.n11\4\Y;.CeJLTC ACIL) C14 :D—J‘Q

hSTIVITY(fi’}x¢)YIJYJ- E£.§ %‘&.ki"

(or: a '15-'12" 7‘ 1: UTIf r :11' c:
. ..-.. 3.1 .' .--. ~-' v Vr LJ - ,. --\- _'

 

as?

Theodore Lynn Rehstock

AN ABSTRACT

SCI; I‘TIU

Submitted to the School of Graduate Studies of Micnigan

State College of Agriculture and Agplied Science

in partial fulfillment of the requirements

for the degree of
MASTER OF SCIJNCS
Department of Chemistry

Year 1951

I," A: j
v F
Aooroved K ‘ 4 /<7’)6((/

Theodore Lynn Reosteck

Recent investigations have ShLBJn that t.ee treat :nent of
red kidney beau plant a with 2,4-diehler0phenexyacetie acid
results in a Me notion of carbehy dretes, increase in nitragen
and amino acids, changes in the vitamin content, and c“ad~ee
in the activity of a number of etzrwws involved in carbo-
hydrate metahnlism (1-5). The pureeee of this investi-
gation wee tn determine if the treatment of red kiiney been
plants with 23L-diehler09heuex=.cetie acid also alterei he
eroteolytic activity. Hemoglobin, 3} .ye ylglycine, cyatinyi-
diglyeine, chlorcacetyl-tyrecine, and eye tinyldi glyr yl-
diglyeine were used as substrates for th.3se dete"“’netiove

Giyrrlfiivci ne ani chloroaee wyl-t reef me were prep er ed by the

I

‘0

methol of Pi char (6), cystinyldiglyeine by the me Jlfld c;
Lorin; and du "vigneaud (7), aud eye tinyldiglycyl i3lysine
by the “ oeeuure of Greenete in (8).

The red kidney been plants were divided into leaf,
stem, and rest tissue. lione cf the tissues showed Garbo oxy-
peptidase or aminOpolypeptidase activity with chloroacetylo
tyrosine and eye nVIdiglycv‘dirlwcire substrates, Peepective-
1y. Bite ptidaee nativity was deteeted in the three groups
of tieeue with glycylglyoine and cystinyldiglycine substrates
Hemoglobin was else hydrclyzed by the red kidney bean plant
tissuee. The proteolytic activity in the leaf tissue of
the treated plants was lower than in the non-treated, the
actirity in the non-treated stems was loner than the treated,
whereas the activity of the treated reet tissue was not

significantly different from that of the non-treated roots.

Evidanca is also pracentad which indicatas that a large
portion cf .43 increase in nitrogen in the treated stem
tissua is 30 percent ethanol insoluble and is probably

'protain.

. ‘. . .47. _-. .1 é» ., _.‘
3313333333 uiuud

l. 3311, 3. 1., R. 3. Luecka, B. 3. Taylor and C. L. ha3n3r.
Changes in chemical comgcsition of the stem3 of red
4133c; L333 plants Lr33433 3153 4,4-343313; '.33u3y-
333313 3311. Fl:ant 9333101., 243219 M9, 1949.

2. wellsr, L. 3., R. w. Lusaka, C. L. Hamnar and H. M.
3341.. unalicsfiiifi iii 3.1331403]. (30:11)le uiCLL U... thifi la‘flVOB
333 r3333 31' red 411333 bean plants traatad with 2,4-
u...Cut.o.UI0:4-LJAUJCJdV5uLc {101610 .iai.t LJSLO-LO, 25326;" 9-

2939 1950.

3. Lu 333, R. 3., C. L. Hanner and H. M. 3311. Effect
cf 2,4-dicnlurophanoxyauetic acid 0n the content of
thiamina, riboflavin, nicotini acid, pantcthenio acid
and cazotu33 in s cams and leaves oi rad kianey 0333
plants. F3.ant. avaio'., 2M; )46-)48, 1949.

4. NealI, w. 9., C. D. 8311, C. L. Hammer and H. I. Sell.
44.333 01 2,4-aiuhlor3phonoxyaowtic acid on the alp3a
333 b3ta awylaas activity in 3113 3t313 and leaves of
r33 413335 bean plants. Science, 111: 2375, 1950.

’ VJ. fig, Go Do 8:33.11, C. L. Ha'flner 8:16.11. ’40 $311.

ct of 9,4-dlcnl rosnanoxvac etic acid on the inver-

, 3p orylase and pectin 33th: xIlasa activity

33 st 3 mg 331 leaves of the ra ad kidtaey bean plants.

113'31010, L)3bCS-)d0, 4.9)00

6. ”13433., E. Enters uchungen user Aminosauren, Poly -
FEthB, und Proteins. Berlin, 346, 424, 1899-1906.

7. r g H. 9. and V. du Vigneaud. Synthaais of

I4tallina 03-41nJ4ulei, 133 and benzy leystein3lglycine

331 .431r 130133103 from clutath .1039. J. .Biol. Chem.,

133JJ" 3‘22, 1 )5.

8. 4.3-3 te.in, J. P. CIstinyl paptides as substrates for
ar‘rmnoiraautidasc and dip entidase. J. 3101. Chem..
124: 255-252. 1938

Th' "\ " I“
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O
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0

3213231.: 3:14.; 3.131;}.- zls’o o o 0
LA? “.3. ~ {Winn o o o o o 0
Materials ani methods

Samples of tissue‘.

Analytlzal methods.
Emy .3313 of Feptldea

\f—

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O O
O O
O O
O O
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131

H" Y WT?“

U JU $123.31

L-Cyatinyldiglycine . . . . . . .

T

Gchylg ycine . o o

Chlorcacetyl-L-tyros

"'ULT3 PJ’L) DISC U38 ICE .

,mafl
3U..- ufoooaooooo

QT")-‘T
__-.;L.LI.LVJTLL.LLY o o o o o o

O O
ine
O O
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d-Cysti LV].di dlycyldiglycine . . .

ACILVO‘JLEDGfiflITS
The author wishes to oXpresa his sincere thanks to
Proteuors C. D. 89.11 and H. M. 8011 for their helpful
guidance and encouragement in the carrying out of this work.
He also extends hie thenke to Profeesor c. L. Hamner for
furnishing the plant materiel. The writer deeply eppreoietee

the financial support from the Rockefeller Foundation.

INTRODUCTION

Only within the last few years have growth-regulating
compounds been used on a large scale. A group of. x lish
workers were the first to make use of euch a compoundxto
control weeds in cats (1). Since that time the demanékfor

the manufacture of such chemicals has resulted in the develOp-
‘-.\

ment of a multimillion dollar chemical business. deco , us
to United States Tariff Commission figures 2,u~dichlorohen-
oxyacetio acid (2,h-D). which is one of a number of grofithe
regulating compounds, was manufactured at a rate of 9l7p000\
pounds in l9h5. By l9h8 this compound was being produc d\ \
at a rate of 27,512,000 pounds annually (2). These figure

illustrate how rapidly the use of these compounds has ohoanded
over a period of Just a few years. h K \\
In the field of plant growth-regulators consideratle H 5‘

‘\.

confusion has arisen as a result of terminology. Thet tFm X

phytohormone or plant hormone has been used in at least two R

“whw

different eenaee. In its restricted sense a hormone isa

_ ‘4

substance produced in any part of the plant where it in u- 3
ences a specific physiological process, and in a broade ‘ 3
sense growth-regulating compounds are generally called Almnt
hormones (3). According to the first definition, the tern
phytohormone cannot be applied to any synthetic substance

which.has not been shown to be present in plants, although

2

the synthetic substance may have physiological activity very
similar to that of a naturally occurring compound. For
example, naphthalencacetic acid could not be classified as
a.hormone3 but indoleacetic acid could be classified as such
by virtue of its being isolated from.plant material. Van
Overbeek has preposed that a phytohormone be defined as an
organic substance. other than the recOgnized energy supplying
substances. which regulates the physiolcgical functions in
plants (3). This definition includes synthetic as well as
naturally occurring substances. vitamins as well as here
mones. and growth inhibitors as well as growth promoters.

The applications of plant growth-regulators are many.
Root initiation can be induced in cuttings of many species
of plants by brief treatment with growth-regulators (A).
By the treatment of some flowers with certain of these
chemicals, parthenccarpic fruit deveIOpment can be induced
(5, 6). Some of the compounds are capable of inducing
develOpment of flower buds and so permit some control of the
time of flowering (7). Other chemicals are capable of in-
hibiting bud develOpment. Alpha-naphthaleneacetic acid, as
well as 2,h-dichlorophenoxyacetic acid. is employed to pre-
vent the preharvest'drOp of fruit (9, 10). The ripening of
bananas is accelerated by eXposure to a low concentration of
2,updichlorophenoxyacetic acid (11). Perhaps the most widely
used of the growth-regulating compounds is the use of 2,h-di-
chlcrOphenoxyacetic acid, hereafter designated as 2,h-D. for

the control of weeds.

Several investigations have been undertaken on the
effect of the treatment of red kidney bean.plants with.2,h-D
on.the metabolism of the plant. It has been found that
there are striking changes in the carbohydrate. nitrOgen,
amino acid, and vitamin contents of the plants following
treatment with 2.h-D (12. 13. 1h). Neely and co-workers
have demonstrated that the activity of several of the en-
symcs involved in carbohydrate metabolism are changed con-
siderably as a result of the treatment of the plant with
this compound (15. 16). In view of the striking changes in
the protein (RX 6.25) and amino acid contents of been plants
following treatment with 2.h—D (12. 13). it appeared to be
of interest to study the effect that treatment with this
compound had upon the proteolytic enzyme activity of red
kidney bean plants.

HISTORICAL REVIEW

The English workers Slade, Templeman, and Sexton (1)
in August, 1940 observed that when alpha—naphthaleneacetic
acid was applied to cats, woody with yellow charlock, at
the rate of 25 pounds per acre, the charlock was killed
with little injury to the cats. To these workers goes the
credit for the first use of a plant growth-regulator as a
selective weed killer. To E. J. Kraus, of the University
of Chicago, goes the credit for first suggesting the use
of plant growth-regulators as herbicides in the United
States. It is reported that Kraus suggested the idea as
early as August, l9hl (17).

Slade, Tomoleman, and Sexton (1) continued their work
by searching for growth-regulators Kore active than alpha-
naphthaleneacetic acid. Cf the many compounds tested, they
found that substituted phenoxyacatic and naphthoxyacetic
acids showed the most promise for selective herbicidal
activity. In 19h1 these workers concluded that 2-methyl-h-
chlorophenoxyacetic acid (Na salt) was one of the two most
active of the compounds tested. Trials in the field in 1943
and l9hh confirmed their findings as to the merits of 2-
nethyl-h-chlorOphenoxyacetic acid as a selective herbicide.

The other most active compound tested by Slade and co-

workers, 2,h-dichlor0phenoxyacetic acid, was further tested

5
by Nutmmn, Thorton, and Quastel (18). Elackman (19) in
19h3 carried out sXpOriments to compare Zdnethyl-hpchloro-
phenoxyacetic acid with 2,k~dichlorcphenoxyacetic acid and
found the former to be more selective and less likely to
injure cross than 2.h-D when applied to control broadleaf
annual weeds growing with cereals.

The first investigators in the United States to demon-
strate the physiological preperties (cell elongation,
morphogenesis, root deveIOpment and parthenocarpy) of sub-
stituted phenoxy acids were Zimmerman and Hitchcock (20).
Hitchell and Banner (21) suggested the possibility of using
2,h-D and similar compounds with ”Carbowax” as selective
herbicides as a result of their search for suitable carriers
for growth-regulating compounds. A short time later in l9hh
Hamner and Tukcy (l7) conducted the first actual cXperiments
designed to test the potentiality of the growth-regulators
as herbicides. Bindweed (Congolvulus srvensis 2,) was
successfully killed by treatment with 2,u-D and 2,u,5-tri-
chlorOphenoxyacetic acid. Since that time numerous workers
have utilized these and similar compounds to determine their
herbicidal effects on other weeds.

Baal (22, 23) observed the telemorphic effects or the
application of substituted phenoxy compounds on sweet pea,
nsrigold and bean plants. The term "telemorphie' response
was used to mean.morph010gical resnonses induced or incited
in a plant even at a point considerably distant from the

point of application of the substance. These effects were

6
manifested by abnormal growth responses of the different
organs or the plant treated with growth-regulators.

As a result of their investigations on the morpholog-
ical and histological changes due to 2,h-D treatment in
bindueed and sowthistle, Tukey.2§ 5; (2h) preposed four
possible mechanisms contributing to the herbicidal action
of 2,h-D: (1) chlorOphyll depletion reducing food synthesis
in tissues, (2) translocation of food interferred with by

phloem proliferation, (3) food reserves depleted by in-
creased respiratory activity, and (h) invasion of soil
organisms due to disorganization and rupture of rhisonic
and root cortex.

Burton (25) studied the formative effects of certain
substituted chlorOphenoxy compounds on bean leaves. It was
found that treatment or young plants with 0.5 percent 2-
chlorOphenoxyacetic acid in carbons: inhibited the formation
of intercellular spaces in the nesOphyll or been leaflets.
Similar application of 0.5 percent h—chlorOphenoxyacetic
acid in lanolin inhibited the activity of plate neristem
in the laminae of bean leaflets. As a result veins were
not distinct and their parenchyna became continuous although
the vascular elements remained discrete. Chlorenchymatous
tissue was confined to the margin. Similar treatment with
0.5 percent 2,h~D in either lanolin or carbons: as the carrier
induced pregressive modification of been leaves. The earliest
leaves were similar to those induced by treatment with 2-chloro-
phenoxyacetic acid. In later leaves the external form and

internal structure resembled that formed in leaflets treated

with the upchloro compound.

Pridham.has reported that seedlings of been plants
sprayed with 2.h-D during ripening of pods developed 2.h-D
syMptmms (virusdike crisp foliage, dwarfing of growth. and
serration and fusion of leaflets) in Juvenile and.mature
leaves (26).

A number of investigations have been undertaken
designed to reveal the mechanism of the action of 2.u-D
on plants. It was found by Mitchell (27) that within.three
weeks after having been sprayed with an aqueous solution
containing 1.000 p.p.m. of 2,h~D, plants of morning glory
were dead and had been essentially depleted of sugars, starch
and dextrin. Soon after treatment sugars had increased
above the amounts in control plants, and then decreased
until nearly depleted.

Rasmussen (28) observed that the reducing sugar content
of dandelion roots increased rapidly following application
of 2,h~D, but later decreased towards the control levels.
The sucrose declined slowly and the dextrin.and levulin
content decreased rapidly after treatment. There was found
an increase in nitrogen soluble in 80 percent alcohol and
also an increase in protein in the roots after treatment.

In experiments employing bindweed, Smith, Hamner and
Carlson (29) found rapid increases in total sugars in all
parts of the plant following 2.h-D treatment; but the in-
ereases were followed by decreases to the control levels
after a few days. The starch-dextrin fraction decreased in

all parts of treated plants. Total nitrogen decreased

steadily in the leaves and increased in both stems and
underground parts. Further evidence of altered metabolism
was found in the increased respiratory activities of the
treated rhizome and root slices.

Workers at the Agriculture Exceriment Station of the
University of Idaho have also observed that 2,h-D treatment
resulted in an increase of the protein content of seven
varieties of wheat (30).

Sell and oowworkers have carried out extensive studies
on changes which take place in red kidney bean plants after
treatment with 2,u-D (12). Both total nitrogen and amino
acids accumulated in greater quantities in the stems of
plants after treatment. The amino acids calculated as
percent of crude protein showed the greatest differences
in the quantity of amino acids of the otom.tioeue in the
case of aspartic acid, lysine, valine, methionine, and
phenylalanine. In View of these results it was suggested
that there is a possibility of the protein being different
in the treated atoms. The reducing and non-reducing sugars
were depleted in the stems of the 2,u-D treated plants. A
considerable reduction in carbohydrate reserves and a de-
crease in acid.hydrolyzable polysaccharides also was observed.
The decrease in carbohydrate content and increase in protein
suggested to these workers that a large portion of the carbo-
hydrate is utilized in protein synthesis. The amount of crude
fiber decreased in the stoma following treatment, and Chb

amount of ether extract increased slightly in the treated

atom tissue.

Similar studies woro carried out on the leaves and
roots of rod kidney bean plants by wallor, Luocke, Hammer
and Sell (13). The leaves of the troatod plants oontainod
slightly lower percentages of nitrogen anfi most of the amino
acids than :1a the control plants. The percentagos of crude
protein and the amino acids valino, histiiina, and arginino
were almost the some in the troatsd and‘untraatad roots.

The other amino acids were olishtly loss in the roots of the
treatad plants. It was suggoatod tnat tho reduction of most
of tho amino atlas 1n the root and loaf tissua may be duo

in part to the translocatlon of those substances to the

Item tissue. A depletion of non-reducing sugars was ob-
served in both the leaves an& roots of the treated plants.
The percentagesof reducing sugars. starch, polysaccharides,
crude fiber, ash, ether extract, unaaponifiablo material,
and fatty acids were practically the some for goth the
leaves and the roots of tho troatod and non-treated plants.

The vitamin content has also been found to change
following treatment of red kidney bean plants with 2.h-D
(1h). The contont of thiamine, rljoflavin. nicotinlo acid
and carotano was obsorvod to be 1333 in the leaves of
treated plants, whereas the content of pantothonio acid
increased in the treated leaves. In tho stems it was found
that the content of thiamine, riboflavin, nicotinic acid and
pantothonio acid increased in the treated plants, whereas the

carotene content was lower in tho treated stems.

10

The marked chemical changes in the treated tissue
suggested that the enzyme systems might be involved. Keely.
Ball, Eamner and Sell (15, 16) carried out studies on a
nwmbcr of the enzymes involved in carbohydrate metabolism.
It was found by these workers that the alpha amylase activity
was considerably lowered and the beta amylase activity
slightly lowered in the treated stem.tiseue. In the leaves
there was a very slight increase in the beta amylase
activity in the treated plants. Invertase activity was not
detected in either the treated or non-treated leaves and
stems of the pea kidney bean plants. Pectin methoxylase
activity increased in both the stems and leaves of treated
plants. Phosphorylase activity decreased in the stem and
leaf tissue of the 2.h-D treated plants.

Hagen, Clagett, and helgeson of the Korth Dakota
Agriculture College have made a study on the effect of
2,h~0 upon the activity of the caster bean 119838 (31).

In experiments in which olive oil was used as substrate and
solvent extracted caster bean meal as the source of enzyme
and various concentrations of the sodium salt of 2.h~D
added, it was found that the lipase activity was inhibited

by the presence of 2,h—D at a very low concentration.

$7."! ",1 17 . V-‘n 0‘
thbtfiIJIcfi 1.2L

Samples of Tissue

of 1 af, stefiL and root tissug. Seeds cf

0

Brooarstic

{.1

the red kidney bean, Phasoolns vvlyaris, were selected for

 

uniformity of size and planted in four-inch pots in the
grosnhousa. Each pot contained four plants which were
treated when the first trifoliats leaves were expanding.

Two replications of 100 plants each were used as a source

of both treated and non-treatsd (control) plant material.
One drop (0.05 ml.) of a 0.1 percent solution of 2,u-di-
chloroohenoxyacotio acid (2,u-J) was saplisd to the base of
the blade of a primary leaf of each treated phant. The
plants wars harvested six days after treatment. At the time
of harvest the sham tissue had proliferated considerably

but there were no signs of nssrosis. Ths'matsrial was dried
in the dark at 30° C. and after drying segregated into leaf,
stem, and root tissue with the hypocotyl, first internods,
and loaf potiolss bsing grouped together as stem tissue.

The material was ground in a micro alloy mill to pass through
a 60-mssh screen and stored in the dark in stoggerad glass

Jars.

12
Analytical Methods
Determination of proteolztic activity with a hemoglobin

ggbstratc. The procedure employed for the determination of
proteolytic activity using a protein as the substrate, with
n few minor alterations, was a modification or the Ayrs-
Andorson method as revised by Miller (32). A one-gram
sample of the powdered olent material was placed in an
sight-inch test tube along with 0.625 g. of Difco Bacte-
Hcmoglobin. The tube was rotated so as to thoroughly mix
the plant material and the substrate. Twenty-five milli-
liters of 0.1 M. acetate buffer of pfi 4.8, which had been
previously brought to the temperature of the water oath.
was added. After agitation and stirring with a glass rod
until a uniform suspension.had been obtained, a trace of
toluene (0.5 ml.) was added. The tube was placed in an
automatic shaking device contained in a constant tempera-
ture water bath held at £80 C. and incubated the desired
length of time. ?cr each determination two tubes of material
were prepared in the same manner and missed in the water
bath at the same time. After fifteen minutes of shaking,
5 ml. of 36 percent t'icbloroecetic acid was added to one
of the tubes and the shaking continued for an additional
five minutes. The contents of the tube were filtered
through paper and the precipitated material washed with a
Infill volume of distilled water. During the washing care
was taken so that the total volume of the filtrate did not
exceed 50 m1. T18 filtrate was diluted to 50 ml. and tho

13
amino nitrogen determined on a 10 ml. aliquot in a Van
Slyke amino nitroaen apparatus. The sample was shaken five
minutes in the reaction chamber of the apparatus.

Five hours after taking the first sample the remaining
tube was treated in the same manner and the amino nitrOgen
determined on a 10 ml. aliquot.‘ The difference between the
two determinations was takon to represent the increase in
amino nitrogen as a result or the action or the protoolytio
enzymes in the plant material on the hemoglobin substrate.
Treated and non-treated tissue were run at the same time for
each determination. The results of the determinations worm
eXpressed on the basis of the increase in milligrams of

amino nitrogen per plant per five hours of hydrolysis.

Determination o£#proteolytic activity using synthetic
substrates. For these determinations 0.750 g. of the appro-
priate powdered plant material was suspended for four hours
in 10 m1. of an aqueous 20 percent glycerol solution. At
thirty.minute intervals the tubes containing the snapension
were tipped several times in order to resuSpend any material
which may have settled to the bottom of the tubes. After
four hours the suspension was centrifuged and the supernatant
liquid filtered. The clear filtrate served as the source

of enzyme. Longer extraction periods did not result in a
measurable increase in enzymatic activity.

The synthetic substrates used for the determinations

consisted of one percent solutions of glycylglycine, chloro-

1’4
scetyl-L-tyrosine, L-cyztinyldiglycine, and L-cystinyldi-
glycyldiglycine.

The nrocedure employed using glycylglycine as a sub-
strate was one by Blngoveshchenskii and Melamed (33). A
series of three tubes were necessary for each determination.
The first tube contained 3 ml. of a one percent solution of
glycylglycino in 0.067 M. ph05pbats buffer pH 7.h and 2 m1.
of the glycerol extrect. The second tube contained 3 ml.
of 0.06? J. phosphate buffer pH 70% and 2 ml. of the glycerol
extract. The last tube contained 3 ml. of a one percent
solution of glycylglycine in 0.067 M. phOSphete buffer pH 7.h
and 2 ml. of a 20 percent glycerol solution. Two-tenths
milliliter of toluene was added to each tube to inhibit
bacterial growth and the tules were incubated in a constant
temnernture water tats at 30° C. for :8 hours. At the end
of this time, the solutions were removed from the rater bath
and diluted to 5 volume of 10 ml. in aliquot of the diluted
solution was analyzed for amino nitrogen in a Van Slyke
amino nitrogen apparatus. The sum of the amino nitrogen
determinations fron the tco blank tubes was subtracted from
the value of the amino nitronen obtained from the tube con-
taining both the nlent extract and the substrate.‘

A similar procedure was emfloyed with the other syn-
hetic substrates. The pentides were dissolved in water,
the pH adjusted to 7 with dilute ammonium hydroxide, and

diluted with distilled water so as to give a one percent

solution. Three tubes were prenared as in the method using

15
glycylglyoine for each substrate. After #3 hours of incu-
bation in the water bath at 339 6., the tubes were removed
and the amino nitrogen determined on all of the tunes with
the exception of those containing cystinyldigiycyldiglycine.

In the case of the determinations with cyetinyldiilycyl-
diglyeine as tn substrate the aolutiene were filtered in
order to cellect any oyetlne that had preeipiteted in the

”I

course at the reaction. lhe preeijitete was dissolved off
of the filter with 6 N. hydrochloric acid and the nitrogen
determined by a semi-micro Ejeluuhl. The tunes centeining
the plant extract enly and the substrate only were treated
in a like manner. In all ef the determinations treated and

non-treated tissue were used at the same time end the results

were exereesed as milligrams of amino nitrogen per plant.

gzpthosia of Pegtidee
L-Gyatinyldiglycino

 

3e321l ehlorofonmato (34}. In a three-necked flask
fitted with a rubber stepper carrying an exit tube and a
delivery tune extending to the oottum was placed 250 g. or
dry toluene. The tunes wereequipped with stepoecks so that
the reaction flask could no disconnected. The flask con-
taining the toluene was weighed and tooled in an ice bath.
Phosgene was bubbled into the toluene until 55 so had been
absorbed. After the required amount of phoegene had been
absorbed, the connection to the phosgene tank was replaced

by a separatory funnel, and with gentle shaking. Sh g. of

16

C. P. benzyl alcohol was added rapidly through the separatory
fennel. The flask was allowed to stand in the ice bath for
one-half hour and at room.temoerature for an additional two
hours. The solution was then concentrated under reduced
pressure at a temperature below 60° C. to remove the major
portion of the toluene, hydrochloric acid, and phosgene.

bicarbobgnzggz:pucystige (3S). Seven and tweetanths
grams of Locystinc were dissolved in 60 ml. of l N. sodium
hydroxide contained in a 500 ml. three-necked flask fitted
with a mechanical stirrer, arousing funnel. and cooled in
an ice bath. Twenty-four grams of the benzyl ohloroformate
prepared above were added drOpwiss to the vigorously stirred
solution while 120 ml. of l N. sodium.nydroxide were added
drOpwise at the some time over a period of 20 to 25 minutes.
The mixture was stirred for en additional 30 minutes and
extracted with ethyl ether. The cooled, aqueous phase was
acidified to the appearance of the blue color of Congo red
with 6 N. hydrochloric acid. The white amorphous precipi-
tate was filtered ahd washed with small portions of cold
water until the washings were free of acid. The dry filter
cake was dissolved in 200 ml. or ethyl ecetete and filtered.
The ethyl acetate was removed under reduced pressure end the
residue taken up in 200 ml. of chloroform. Dicerbobenzoxy-
L-cystine separated as fine needles, mhp. 122-12h9 O.

bicarbobenzoxzéL-cgatinlldlolzcine (36.37). Ten grams
of finely pulverized dicsrbobenzoxydL-cyetine suspended in

60 ml. of anhydrous ethyl ether in s 125 ml. glass stopperod

i

17
O

Erlenmeyer flask was cooled in an ice-salt bath. Si3h
and two-tenths grams of finely pulverized phosohorous penta-
chloride were added and the suspension shaken vL Nor us 1y.
After several. moot es the material we nt int 9 solution. The
stepper was removed.momeutarily at frequent intervals in
order to reloaee any pressure which may have built up in-
side the flask. After 15 to 20 minutes with shaking and
cooling, the aid chlorY e of cicaroojui oxy-L-cvstine
precL i.ated. An additionfl 10 to 15 ml. of dry etth
other was add ed a: id the material filtered on a dry filter.
A’ter washing the precipitate with a Stall volm etc of dry
ethyl other. the pro d” ct was added in small portions along
with 38 ml. of l N. sodium hydroxide to h.d g. of glycine
dissolved in 50 ml. of l N. sodium hydroxide cooled in an
ice-salt bath. During the addition of the acid chloride
and for one-half hour afterward, the solution was stirred
vigorously. The cooled alkaline solution was acidified with
hydrochloric acid and the crude preoigitate of dicarbo-
beneoxy-L-cystinyldiglycine filtered. The amorphous pre-
cipita to was washed several t..mee with cold water unti
thew achin2s wsza neutral or only rain uly aoir to Ccn2o red.
The moist solid was dissolved in 153 ml. of boiling dioxane
and the hot solution filtered. The filtrate was evaporated
unoer reduced pressure to a voloieo of (5 ml. and on standing
fine needles of d‘OanOJad”“KJ~u- t.n,l.i lyoins aJpeerod.
Those were filtered off and washed with ether. After extrac-

.tion with ethyl acetate and crystallization the melting

18

point was 175-1?8° C.

Lgcgstingldirlyoing (37). One and seven-tenths grams
of sodium out in small pieces was added gradually to 6.2 g.
of dioarbobenzoxy-L-oystinyldialyoine dissolved in 50 ml.
of liquid ammonia contained in a 250 ml. three-necked flask
fitted with a mercury-sealed stirring device. The solution
was cooled in a trichloroethyleno-solid carbon dioxide bath
and stirred gently. The completion of the reaction was
shown by the annearance of the blue color due to free sodium.
After the ammonia had been allowed to evagorate, the flask
was evacuated on the water pump in order to remove the
remaining ammonia; and the residue remaining was dissolved
in a small volume of water. The flask was again evacuated
for a short time and the cooled solution made neutral with
hydriodic acid. The reduced peptide was oxidized by aerating
the solution until the sodium nitro-prusside test was neg-
ative. The solution was made slightly acid to litmus with
hydrioaic acid, concentrated in vacuo until crystals of
sodium iodide began to separate, and the oxidized peptide
precipitated by the addition of several volumes of ethanol.
The comoound was obtained halogen free by repeated solution
of the amorphous product in a small volume of water and
precipitation with ethanol. The amorphous compound was
then dissolved in a small volume of water (15 ml.), enough
ethanol added to give the solution a cloudy appearance. and
the mixture allowed to stand overnight in the refrigerator.

The peptide first appeared as a syrup but gradually solid-

19
ified. The peptide Was filtered, enough ethanol was added
to give a cloudy aopoaranoe, ani the cooling repeated. Tho
melting point of the crystalline groiact was EDA-236° C.
(decomposition).

3? h

A2131. Cale-:1. for lejiilg-Eé’fliggg' “Liz-'3: n, 14.)). ound:

N. 14.29.

L-Cystinyldiglyoyldiglyoine

310a oooonzoxy-L-ggstioildiilgqxldiallfilna (33).

.__.4 -__.-

’3

 

Twenty grams of glycine anhydrico was dissolved in 100 m1.
of 2 M. sodium hydroxide and the aolution allowed to stand
for one-half hour at room temperature. An equal volume of
water was added and the solution chilled. With vigorous
stirring, dicarbobenzoxy-L-oyetinyldichloriae. prepared
from 23.2 g. of dioaroobenzoiyéL-oystine as previously
described, was added in small portions along with.the alter-
nate addition of a total volume of 80 ml. portions of l N.
sodium hydroxide. The addition required about an hour and
the stirring was continued for an additional half hour.

The solution was filtered and the cooled filtrate treated
with S N. hydrochloric acid to the appearance of the blue
color of Congo red. The compound appeared as a gelatinous
mass which was immediately filtered off and washed with cold
water. The product was dissolved in 150 ml. of hot dioxane
and evaporated to 50 ml. under reduced pressure. On the
addition of the dioxane solution to a double layer of ethyl
ether and water, an oil separated which rapialy crystallized

into needles on standing in the refrigerator. The material

20
was filtered, washed with cold water, other, and dried.
After extraction with hot ethyl acetate, the product, a
white solid, melted at 207-239° c.

Lyczstinyldiglzozldialycine {38). Twelve and six-
tenths grams of dicarbobenzoxy-L-cystinyldiglycyldiglycinc
was dissolved in 250 ml. of liquid ammonia. The three-
neched 530 ml. round bottom flask containing the solution
was fitted with a mercury-sealed stirrer and cooled in a
trichloroethylene-dry ice mixture. Metallic sodium was
added in small pieces until a blue color appeared in the
solution. After the ammonia had been allowed to evaporate,
the flask was evacuated on the water pump for several hours.
The residue was dissolved in enough 0.5 H. sulfuric acid to
bring the reaction Just acid to litmus and treated with a
slight excess of Hookin's mercuric sulfate reagent. After
standing several hours, the mercury salt was centrifuged
off and washed five times with distilled water, snapcnded
in water, and treated with hydrogen so ride gas. The filtrate
after aeration was again treated with the mercuric sulfate
reagent, centrifuged, and washed. After removal of the
mercury with hydrogen sulfide, the filtrate was aerated and
treated with saturated bar um hydroxide solution until
weakly alkaline. The cerium sulfate was centrifuged off
and the supernatact solution treated with a rapid current
of air. when the oxidation was completed as shown by a
negative sodium nitroorusside test, the solution was shaken

with norit for one-half hour; and the clear, colorless

21
filtrate was treated with enough dilute sulfuric acid to
exactly remove the barium hydroxide. after removal of the
barium sulfate by centrifugation, the supernatant solution
was evaporated to a small volume (15 ml.) under reduced
pressure and treated with a large sxcoss of ethanol. A
colorless oil asparated which rapidly crystallised in the
refrigerator. After the crystallization was repeated tnrec
times, the oroiact was dried in a vacuum desiccator GVcr
concentrated sulfuric acid. ins melting point of the white
crystalline oroiuct was 96° C. (fiecoaoosition).

Anal. 351131. for Clgflgaonfléflz'a £5.20: N, 16.7. round:

I‘:. 16.70

Glycylglycine

Glxcigananhxdride (39). A mixture containing 70 g. of
glycine and 350 ml. of cthylena glycol was heated with con-
tinuous stirring tor 50 minutes at a temperature of 174° to
176° 6.; and the dark, brown mixture was cooled overnight in
the refrigerator. The suspension was centrifuged and the
brown, crystalline mass washed with a small volume of
absolute methanol until the washings were nearly colorless.
‘The brown crystals were dissolved in 220 ml. of boiling
water and the solution cooled overnight. The crystals were
filtered off. washed with methanol, and air dried. The
product was dissolved in 250 ml. of boiling water. docolor-

ized with decolorizing carbon, and the hot solution filtered.

The crystals, which formed upon standing overnight in the

22

refrigerator. were filtered off. wadhad in turn with ice-
water, 50 percent methanol, and aoaoluto methanol. The
product was pure White crystals of 2,5-d1kotop1pw azizle.

Glyoylglyoino (no). Ono gram of pulverized glycino

 

anhydrido was shaken with 10 ml. of l‘N. sodium hydroxide

at room temperature. The 5lyoino anhydrida wont quickly
into solution; and after 15 to 20 minutes, the solution was
neutralized with an equivalont amount of l N. hrorocxlowfo
acid. The solution was evaporated molar rolueed 3M3331fa to
1 Email volume and ooolod. Tho glyoylglyoina aopoarod a3 a
colorless crystalline mass. Holtin5 ooint 209° C.

Ana]... C910d. for 6&3603312'1120: ‘1, 13.6,). FOEUId: 3?, 18007.

Chloroaootyl-L-Tyrooino (Al)

len grams of -ty vroslne was suasondod in 70 ml. of
absolute ethanol an d hydro.3 on ohlorid o 5&5 bubbled tlzrw 5b.
the sus)o sion until solution was complete. One hundred
and forty millilitors or aosoluto ethanol was added and the
aolution refluxed on the stoam boon for 24 hours. Aftsr
this time the solution was evaporated to dryness under
reduced pressure.

Ten grams of the crude hydrochloride of the ethyl
ester of tyrooine was covered witn 160 ml. of chloroform.
After cooling to 6° C., bl ml. of l E. sodium hydroxide
was addod with shaking. Tho free ester quickly went into
solution in tho chloroform. Five grams of ohloroaoetyl
chlorldo,dissolved in 50 m1. of chloroform. was added in

small portions to the cool mixturo. After about one-half

23
of the acid ohloridé had been added, 20 ml. of sodium car-
bonato solution (U07 5. of the anhydrous salt) was added
alternately with shaking to the cooled mixture. At the
close of the reaction, the chloroform phase was separated
and dried with 2 g. of anhydrous sodium sulfate. Host of
the chloroform was removed on the steam bath and the chloro-

ootyl-L-tyroaine ester crystallized from the solution upon
the addition of a small volume of petroloum other.

Ton grams or the ethyl ester of chloroaootyl-L-tyrosina

was dissolved in 70 ml. of l N. sodium.hyfiroxido. After
15 minutes the solution was nontralized with hydroohlorio
acid and in a short time the chloroacetyl-L-tyroaino began
to crystallize. The crystallization was completed on
standing overnight in the refrigerator. Melting point
153° to 155° 0. '

Anal. Calcd. for CllHIZQHECI‘ N, S.hh. Found: N. 5.65.

RESULTS AND DISCUSSION

The results of the determinations of proteolytic
activity are summarized in Tables 1 to h.

The preteolytio activity witn a hemoglobin substrate
is shown in Table l. The activity in the 2,4-D treated
stem tissue is almost a third more teen in the non-treated
tissue. neterminetions with the leaf tissue snow the
reverse trend. In this case, the leaves of the non-treated
plants show almost double the proteolytio activity of the
treated leaves. Root tissue shows a very slight decrease
in the activity of treated tissue. The results as deter-
mined by the metnod of Van Slyke after five hours' hydro-
lysis at £30 C. are expressed as the increase in milligrams
of amino nitrogen per plant. In experiments in which the
temperature of the reaction and the p5 of the buffer we:
varied, it was found that the maximum.hydrolysis for five
hours took place at h8° C. and pH h.8. In order to be
certain that all of the increase in amino nitroxen was a
result of enzymatic hydrolysis of the hemoglooin substrate,
determinations were carried out on separate samples con-
taining the substrate only and the plant material only. In
neither of these cases was there a significant inorease in

amino nitrogen in the course of five nours under the cen-

TABLE 1
I'Jf‘I‘ 01‘“ 2.4-1):mimicvizlmoxmc5'21C ACID 0?: E3

PRO-‘i‘iiifi-LYTIC AC"{T‘IVITY 02.9 mg.- s=13,.i.~'2_, Lei}? mm .1100? T133322;
OF Tag R 1a 1:133:33: 3.15m 3133?: U 312m A amaziosxs SUBSTRATE“

 

Nonctreated Treated

 

 

Tissue regiieatgiwfi __ replicate~ ‘_
1 2 1 2
Stems 1.73 1.39 2.50 2.h8
Leaves 1.62 1.93 0.96 0.94
Roots 1.02 1.05 - 0.38 0.9h

vv w

sEXpressed as the ineresso in gilligrams of amino nitrogen *
per plant for five hours or reaction at h3° C. and pH h.8.
ditions of the eXperiment, which indicates that the increase

in amino nitrOgen in the determinations was a result of the
enzymatic hydrolysis of the hemoglobin substrate.
Table 2 shows the results of an eXperiment in which
the proteolytio activity was determined on the stem tissue
employing synthetic peptide substrates. With glycylglycine
the treated stems have more than double the activity of the
nonotreatsd material. The same trend is onserved with L-
eystinyldislycine. 'When the substrates were either L-
cystinyldiflycyldiglyclne or chloroacetyl-L—tyrosine, ne
evidence of enzymatic hydrolysis of these compounds was noted.
The results of the determination of proteolytic
activity with leaf tissue and synthetic substrates are
summarized in Table 3. The treated leaves show less than
half the adtivity of the non-treated leaf tissue with glycyl-

glycine. With cystinyldiglycine the activity of the non-g

if.“ .5". 1
m; 050

‘1‘??? {P157
.I. 3.

"1
£1.31.) 1.1.5“

CF, 2 . 2+-.L:)IC

‘2 Lg. ._;..£'L 4?“

TABLE 2

L

riL 01:0 115-350 {MC
’T’I‘II} ACTIVITY O17‘I1Il1

,9
5.153411 V'jf‘t’J/V :v'.‘ ‘.“.
?LJL&Ii ' ..»J.. .l\ .3 .J 3-): '

3II3 LCIQ C"

 

W... ‘1 *
”Mm—w

Substrate

 

 

THE

 

26

 

 

;W 71%? {W “dEZ'
11-41111 SU”?'Z:‘A"‘3S*
Non-treat? d Treated
replicate replies4
1 2 1 2

 

Glyoylglycine

L-Cystinyldiglycine
L-Cystinyldiglycyliiglycine

Chloroe ieetyl-U-tyrosine

0.22
0.5t
0.00
0.00

A“

A

0.00

9.50

1.62,

0.50
0.00

 

1113‘"? ‘ CT

' “ II “it .1.' '
1 :91
4. J...) 'Jtl ..

6 increase in milligrams of amino nitr03en
43 hours of reaction at 30°

03.5.1313 13.1 3

T3”
an ME?
331 vs

 

 

Substrate

2,h-DTCHLGIO?*4%UIIAULTIC A“
P?)- "JLII"" ACTH THY (‘7 IJI": T177. ‘1' c:-
n" EEK" PLfiIIT USIHV’Sli ELIE”
Non~treated
replicate

Glycylglycins
L-Cystinyl d1

Treated

rGUliCHt

 

l

0.20

aglycine 0037

2

0.21
0.28
0.00

,0.00

1

w

0.13
0.13
0.00

9.00

5*
0.13
0.10

0.00
0.00

 

L-Cystinyldiglycyldiglycine 0.00
Cnloroacetyl-L-tyrosine 0.80

anresSeu as
per plant ior

hB hours of reaction

the increase in milligrams of amino nitro;en
a1. 300 Co

27

treated leaves 13 no 6 than doubla the activity of the 2.4-0
treabad mat 3r121.1 AS'was the caaa with tha stem tissue. the
leaves did not Show any hydrolytic activity on the substrates
cystk @131 ycyidiglyoine or enlaroacetyl t.fi 05 1:6.

Table A shows that tha nan-trnated root tiasua has
slightly, but probably nut 3131 III 0&nt1y, mora activity
than the Wreatwd tissue when the substrates are a ycylglycine
and cystinyldiflycine. Again as was observed from the
results of exneriments with stem and laaf tissue, the root
tissue was nut able to bring about the hydrolysis of cystinyl-

d1 glycyliiglyoine uni chloroacetyl tyrosine.

 

 

 

 

 

IA LE 11
EFTECT {WT 2,h—u1vuu13333:"“”LCITIJ 1310 GT TnE
PRGTCQLYfilC ASWLIVII‘I OF THE RQC‘I T ”“UL C” THE
REID 3313111-? 5:14.12); I‘LAIH USES-111$; SII‘Z'I14111‘IC ”131,1.111’11 133*
Ron-treatsd Treated
Substrate replicate replicate;
1 2 1 2
L-Cystinyldibljcine 0.70 0.82 0.63 0.53
L-Cyatinyldidlycyldiglycine 0.00 0.00 0.00 0.00
Chloroacetyl-L-tyrosina ' 0.00 0.00 0.00 0.00

*Exorasaad as the increase in‘millis ;rama of amino nitrovan
per plant 10? M3 hours of reaction at 30° 0.

Specific substrates for aminOpolypeptidasa, carboxy-
peptidasa and dipeptidase hava frequantly been. reSpectively,

lauoyldiglyc inc, ohloroaoetyltyrosine and leuc; lulycine. Tho

2%
methods for estimating the hydrolysis of these compounds
have often times employed a titration procedure. In the
event of the determination of aminooolypeptidase activity
in a biological system in which a dipeptidace is also present,
leucyldiglycine will not be a very satisfactory substrate
since the glycylglyoine, which is Split off by the amino-
polypeptidase, will be further split by the dipeptideso.

As a result, a titration procedure will not give a true
indication of sminOpolypeptidsse activity but a combination
of aminOpolypeptidase and dipeptidase activity.' Creenstoin
(38) suggests as a substrate for sninopolypeptidsse a tri-
peptide containing a very insoluolc amino acid in the acyl
position. The amino acid can be filtered off and determined
separately from he other products of hydrolysis. Exocri-
ments have been resorted by Greenstein (33) which show that
cystinyldigljcyldiglycine is a satisfactory substrate for
determining aminOpolypeptidase activity in the presence of
carboxypeptidase and dipeptidssco

The determination: of amino nitrOgen in this work war.
done in a Van Slykc amino nitrOgen apparatus. A titration
procedure was not convenient to use because of the presence;
of colored substances in the plant extracts which made the
and point of the titration difficult to detect.

It has been reported that the amino acids glycine and
cystine give nitrogen values with the Van Slyke method of
analysis which are slightly higher than the theoretical (h2).

In the case of cystinc in the determinations reported in

29
this work. the error is eliminated when the amino nitrogen
value of the blanks is subtracted from the value of the
reaction tube. The major portion of the error due to glycine
will also be eliminated for the cams reason. The small
error remaining will not be large enough to significantly
alter the results.

Glycylglycine and cystinyldiglyoine are substrates
used for the detection of dipeptidase activity. Chloro-
cetyltyrosine is a specific substrate for carhoxypeptidase

activity and cystinyldiglycyldiglycine is specific for

G

aminOpolypeptidase activity (33). Evidently there were no
enzymes present in the immature bean plants which were able
to exhibit oarboxypeptidase or aminopolypeptidase activity;
or if there were such enzymes present, they were not able

to hydrolyze the substrates employed in these exocriments
for their determination. oipeptidase activity was indicated
in both treated and non-treated bean tissue. The proteolytic
activity determined with either hemoglobin, glycylglycinc

or cystinyliiglycine as the substrates slowed the same trend
in the 2,h~D treated tissue and of soproximately the same
relative difference.

The decreased proteolytic activity in the 2,h-D treated
leaf tissue was to he expected since there was evidence of
an inhibition of the growth of the leaves of the treated
plants. although the total over-all dry weight of the
treated ana non-treated plants was about the same, the weight

of the leaves of the treated plants was less than the weight

30
of the non-treated leaves. Sines the weight of the stem
tissue of the treated plants was greater than the weight
of the stems of the non-treated slants, the total weight of
the two groups or plants remained anoreximately the sanef
Weller, Luecke, hamner and 8811 have shown that the leaves
of 2.3-3 treated plants contained lower percentages of
protein (XX 6.25) and amino acids than did the leaves of
the non-treated plants (13). These conditions might be
cupccted to result in lower protcolytis activity in the
leaves of the treated olants.

The results of the determinations of protsolytic activity
in the stsm tissue show that the sctisity is increased in
the treated plants. This increased protcolytic activity
indicates the possibility of several abnormal conditions
occurring in the treated stem tissue. If it is assumed
that the proteins of the plant are in a state of steady
breakdown and resynthcsis. the increased proteclytic activity
could be taken to indicate that there is an accumulation
of protein in the stems of the treated plants. Sell. Luecko.
Taylor and flamncr have shown that there is a considerablo
reduction in the carbohydrate content of the stems of
treated red kidney bean plants together with an increase
in nitrogen and amino acids and suggested at this tine that
the reduced carbohydrate content is due to a utilization
of these compounds for the synthesis of protein (12). The
possibility exists also that the metabolism of carbohydrate-

is altered in some way and as a result the protein can not

31
be further utilized and tends to accumulate.

Th3 possibility arises also that the increase& protec-
lytio actiTity results in an ac'amulation of amino acids
an& other pr otain dagradation products in the traated stam
tissue. In an éttanpt to clarify these possibilities, an
orderiwa1t was findertaken on the stam tissue to separata
and date rmins .?13 protein and non-nrctein nitro; an of the
2.3-3 treatac and non-treated tiaaua. The plant material
was extra ctad with 30 percent etrxanol and the nitrogen was
determined in tha ethane l extract.- The nitr0g3n remaining
in tha regiduo was taken to be due primarily to protein.
The nitr01en soluble in the 50 percent ethancl was assumad
to be primarily €15 to amino acids and other soluble products
0f protein degraaatien. The rasults of this experlman

are 3.1«arizvd in Table 5.

 

 

 

 

 

T11; ELE S
LIII”! 01 2 h-“ICHLPIF” axoxyacawzc “CID C-N 11: 1
571 TI. RID-1': {-110— ‘..I MK) “$1....2" C’ J 1:1..2. .3: ._-. flavufial‘ 53‘? ii 123;" KI. 435.1:3. ‘5!"K“ " .1211?“ 1,7123
(Exgressad on 3 d1 3 w.-.at basis)
Non-treated Treated
raplicata replicate
Constituent 1 2 - 1 2“
ii # Percent
Total n1trozan 3.02 3.2h n.61 5.00
Soluble nitrogsn 1.Kk 1.55 1.82 2.10
(50 pa 39 nt aethanol)
Insclublo nitrogen 1.58 1.69 2.79 2.90

*111 nitéc gen determined by ’jeld 111 method

32

The results of this experiment indicate that the bulk
of the increased nitrogen content of the treated stems
appears to be located in the protein fraction, although
there is an indication that a small portion of the increase
in nitrogen is due to amino acids and other nitrogenous
products. The character of the protein in the treated scam
tissue comgarod to the non-treated tissue is not known at
the present time. The work of Soil, Luooko, Taylor and Hamner
(12) on the amino acid composition of the troated.ana non-
treated atom tissue indicates that the composition of the
protein in tho treated tissue in diffsrent from that of the
protein in the non-treated tissue.

The differences in the protoolytic activity between
the treated and non-troatod root tissue was not largo

enough to be of significance.

SUMflARY

l. Tho protoolytic activity of tho loaf, atom and
root tissue of red kidney bean plants treated with ',u—dl-
chlorOphonozyacctic acid and non-treated plants was deter-
mined using homoglobin, glycylglgcinc, L-cystinyldiglyoinc,
L-cystinylaiglycyldiglycinc and chloroacstyl-L-tjrosino as
tho suostratos.

2. With hemoglobin, glycylglycino ani L-cyStinyldi-
glycine as the substrabos, the protoolytio activity of the
non-treatad leaves was almost double that of the treated
leaves. The activity of the treated stoma was almost double
tho activity of the non-troatod atoms and tho proteolytio
activity of tho treated roots was slifihtly loss than tho
non-treatcd roots.

3. Loaf;stom and root tiscua did not hydrolyze chloro-
acetyl-R-tyrosino and L-oystinyldiglycyldiglyoino.

A. When the nitrogen components of the treated and
non-treated atom tissue wcro partially fractionated with
80 percent ethanol, the major portion of tho increased
nitrOgcn content of the treated stoma was found in the

insoluble (protein) fraction.

1.

3.

h.

S.

6.

7.

9.

10.

11.

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