An apparent seleniferous leaf wax from Stanleya bipinnata

Vol.

11, No.

1, 1963

BIOCHEMICAL

AN APPARENT R. Division

of

Received

The parent of

the

pathways

(a

McColloch,

25,

J.

of

It

as

does

as

methionine,

In

the

member

which

was

front

during

has

for

Se are

been

COMMUNICATIONS

STANLEYA

and

S.

of

BIPINNATA*

K.

Brown

Wyoming,

mustard

Laramie,

soluble

in

Wyoming

counter-current

and

organic

form

Se follows in

and

their

Beath,

I&g).

(Beath

and

the

same

ap-

metabolic

sulfur-containing

compounds

1958). organic

Se-compounds

a fraction

organic

(Trelease

sulfur

(Shrift,

1 y),

plants

that

for

various

indicator

in

is

assumed

etc.

fami

known

plants

substitutes

studying

certain

well

generally

and

of

by

Stanleya

cysteine,

the

very

in

sulfur

course of

Hamilton

(Se)

Se accumulated .

WAX FROM

University

selenium

requirement

1947)

W.

RESEARCH

1963

accumulation

Epson,

such

J.

BIOPHYSICAL

LEAF

Biochemistry,

obligate

All

SELENIFEROUS

Agricultural

February

AND

was

solvents

and

distribution

found

which

between

in

Stanleya

in

various

moved

close

ethyl

ether

bipinnata

preparations to and

the

ether

0.05N

aqueous

HCI . This

fraction

measurement

by

order

to

Se75

into

were

transplanted

of

the

label

the

the

obtained

modified

facilitate

in method

Se detection plants.

large

survived.

watering

at

intervals

two

months

of

amounts (Klein,

and visible with

and

from grown growth

selenium

1947)

measurement

plants,

pots When

small

and

Stanleya to

plants by

was

in

it

was

not

was

decided

the

field

the

greenhouse.

was

evident

a solution

of

Se

individual

leaves

detection satisfactory. to

(Shirley

In incorporate

Basin,

Wyoming),

Approximately they

plus

and

Se

were 75

10%

given

in

the

a Se form

of

H2Se03. After plants

*

and

Pub1 lshed Station,

about separately

extracted

growth, by

macerating

with approval of the Director, as Journal Paper No. 201.

7

with

Wyoming

were different

Agricultural

cut

from

the

non-polar

labeled organic

Experiment

75

Vol.

11, No.

1, 1963

sol vents.

The

evaluate

the

relative

purity

the

BIOCHEMICAL

radioactivity

and

efficiency

of

of

extracts

and

AND

the

infrared

each

to

were

were

extract

obtained.

residues

RESEARCH

spectra

solvent

extracts

the

BIOPHYSICAL

pelleted

in

run

were

KBr

on

75 and

Se

Solvents

COMMUNICATIONS

and

the

extracts

determine

to the

then

evaporated

from

infrared

spectra

again

taken. At it

was

at

13.75

point,

noted

gested was

this

that

and

minent

the

in

and

The

free 13.95

all

the

any

degree

showed

peaks

purity, doublet

spectra.

chains along

of

a prominent

solvent-run

hydrocarbon

information,

F

spectrum

acid

and

of

whose

with

suggested

Stanleya.

not

been

This

sug-

transition

the

presence

that

the

point of

pro-

extracts

were

Stanleva

the

by

5% to

infrared

a number In

leaves

10%

Upon

and

dipping

whole

F rinse.’

KBr,

amount

‘eaves,

rinsed

from

fair

of

the

spectra

were

fairly

of

indiv’dual

Skelly

were total

evaporating

in

obtained

30

F for

then leaf

the

pure

counted

with

selenium

had

Skelly which

F and were

typical

waxes.

ester

in

carbonyl of

imply Howeve

completely

Subsequent of harvested assumed.

In

rinsed

and

Skelly in

characteristic

in

the

varying

shown

data

present

individually

rinses

the

residues

be of

and

into

The

should

an amount

spectra

These

I

that

the

the

long

indicate

samples

absorption

surfaces

Skelly

extracted

pelleting

waxes

harvested

result

of

in

This

not

pelleted

lacking

carbonyl

external

were

been

was contained

ester

that

the

seconds. the

which

of

did

waxes.

on

leaves

spectra

spectra

temperature.

Reasoning form

the

the

samples

room

acid

rich

all

13.95,

that above

although

high

that r,

proof

Figure

’

contains

absorptions,

and

molecular

weight

a significant of

this

the the

hydrocarbon

prominent

doublet

absorptfons, at

13.75

is

produced

hydrocarbons.

amount has

expected

been

of beset

seleniferous with

wax

difficulties

and

successful.

ratios plants

of

rinsabl e count to are lower; therefore,

8

total no

I eaf count “typical”

on new regrowth values can be

has

and

Vol. 11, No. 1, 1963

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

‘i 60. 5 ; 404-l 5

20-

; 0.

0, I

3

2

4

5

I

I

I

6

7

8

wave1 eng th Fig.

1.

Infrared spectrum 1 eaf extract .

Specific the

loss

the

method

is

activity

of

60% of

believed

using

could

to

90%

of

(Klein, to

the

of

test

potassium

not

be

have

been

tube

method

determined

this

by

of in

the

sample

digestion

was

according

to

by clearing

Furthermore, active with

the material

All

ty

to of

of

Se was

CSZ’ should

is

al cohol rule

In

during

various

Skelly

the

radioactivity not

through

an

because

digestion,

with

perchloric

method,

it of

the

possible

to

digestion.

When

visibly

complete,

been

oxidized,

asbestos

material acid,

was

had

mat

of

using

of

appeared

yet

F Stanleya

samples

stages

(and

of

filtered

this

method

apparently

out

the

determine

obtained,

the lost

and

metallic

leaves to

amount

after Se,

with

the

pelleting which

lost.

since

the

radio-

after

treatment

Se

is

dissolved

soluble

9

of

the

wax

sacrificed activity.

pre-

in

an

Although

higher

than

the

a

reliabil

significance.

the

in is

F was

slightly

little

wax

most

specific

only

of

and

Skelly

evaluate

has

lability when

harvested

was

therefore,

of

rapidly

was

Se

the

and,

s,

material

rinsing

evidence

Se75 and

by

titration

Further

sample

present

digestion

the

the

a characteristic

1961).

25%

Se was

plant

chemically

the

tracer

the

remaining

obtained

attempt

sample),

14

sulfate.

the

paration

the

remaining in

hydrazine

trace

of

the

of

during

is

loss

(Feigl,

radioactivity

13

12

pellet

on

attempting

determine

judged

II

(radioactivity)

That

shown

complete,

bromide

selenium

1947).

lo

(microns)

the

the

I

9

in

the

in

Loss of

K8r. and

wax

stable

preparation

chlorinated count in

from this

is

that

solvents, CS2 solvent.

solutions

i-

BIOCHEMICAL

Vol. 11, No. 1, 1963 Infrared This

group. I ies

is

4.25~

at

lie

spectra

beyond

not

CO2

15~

Se bond

not

yet

surprising,

where

the

organic

have

AND BIOPHYSICAL been

since

interferes;

range

of

absorptions

helpful

in

R-Se-H

instrument

are

not

known

locating

a functional

absorption

R-Se-R

our

RESEARCH COMMUNICATIONS

is

and

R-Se-Se-R

and

are

with

weak

Se

and

theoretically

absorptions

also

weak.

sufficient

theoretically

Other

possible

certainty

to

be

of

much

assistance. The belief

results that

excel

Se

lent

the

hydrocarbon

formation

two

stays

up

saturated

clathrates

with

the

SO:50

three

times

formed

by

saturated

the adding

the

MeOH The

and

washed

by

water-insoluble

waxy

was

filtered

volume

of

HeOH.

only the

were

material

in

above

up

in

Washed

cl athrate

Washings

from

Aqueous phase cl athrate Hexane

soluble cl athrate

*

Label

wax

tenacity

is

chemically types was

equal

clathrate and The

to by

an

al lowed

to

crystal

times

unstable

only hot

MeOH

10

was of

with

water

urea-

benzene. and

the

F.

cl athrate sample

Benzene washed counts/m/total

2,147 33

108*

0

decomposed

in

I ize

with

volume

decomposed

from

taking

cl athrate

washing with

by urea-

equal

2,163

in

clathrate

of

three

whi ch

bound

formed

benzene-washed

benzene)

forms

volume

washed

our

with

of

503

497i’i is

an

wax

the

two

to

I.

696

from

of

The

clathrate to

the

Skelly

MeOH washed counts/m/total

cl athrate

it

results

decomposed

TABLE

Fraction

and

procedure

subsequently taken

1956)) that

it

it

repeating

clathrates

adding and

(dissolved

molecules.

washed

warmed

which

wax

MeOH and

precipitate’s

the

evidence

wax

indicates

I gives

was

after

circumstantial

Stanleya

indicated

mixture

temperature,

of

clathrate

MeOH-Benzene

The

strong

(Trutter,

Table The

MeOH.

room

a part

molecules.

in

lend

urea

experiments.

wax

experiments

chemically 1 ine

label

Se75

at

is

cystal

the

the

of

cl athrate sample

Vol.

11, No.

1, 1963

From formation

BIOCHEMICAL

the

we1 l-known

described

high

molecular

chemi

call

were

no

by

different

Chromatography

of

microadaptation in

of

terms

with

of

loss

the

a seleniferous

the

1% ethyl

fraction had contain

sterol

ability

to

tuents

form

of

Skelly

esters,

carotenoids,

urea

and

infrared wax

with

free-acid

IV

showed

fraction

the

fraction

from

gel

the

(Hi rsch

and

of

the

a

clathrates

we were

in

the II

by

MeOH

and

which

III, I

waxes.

sterol

esters

U.V.

and

in

absolute

fraction

vegetable

contain

dealing eluted

1958),

eliminates out

1961)

destructive

fractions

and

I should

proved

eluted

in

(Lis,

radioactivity

Ahrens,

ruled

the

that

22%

eluted

by

1958)

hydrocarbons,

absorption

indicated

did only

should

The as

consti-

visible

hydrocarbons

in

new

predominantly unreliable I had

in

fraction hydrocarbon

a Se-to-dry-matter

fatty

IV.

the

acid.

of

IV

spectral

and/or

or

ester fatty

5.74

carbonyl

and

resulted

but

10%

infrared

spectra

ester

and

fraction

IV as

of

11

a hydroslight

phospholipid,

spectrum spectrum

the

ratio

only

contain

infrared

Again,

of

and

infrared

I I,

determination

of

of of

the

fraction

spectrum

of

acid.

fraction

fraction

spectrum

should

The The

free

of new

at

this.

acid

a portion

expected

Fraction

support

amounts

of

chemical

not

the

absorption

5.84.

neither

large

radioactivity

I was

carbonyl

free

but

I I I showed

fraction

at

spectrum

hydrocarbon,

the

of ester

Re-chromatography

on

and

was

fraction

prominent

infrared

fraction

I as

silica

70%

are

with

containing

recovered

evidence

(1)

carotenoids

clathrate

consistent

material

Ahrens,

practically

spectrum

carbonyl

the

appeared

I are

urea

waxes.

carbon

of

and

matter

clathrates I,

on

further

to

Table

wax

procedure,

dry

of

of

1,

wax

F fraction

the

the

(Hirsch

this

Therefore,

The

85%

In

of

data

Figure

furnished

According

fraction

vegetable

II

but

of

COMMUNICATIONS

specificities

hydrocarbon

of

of

count.

determinations.

but

seleniferous

method

in Most

negligible

the

wax.

ether

(IV).

spectrum

Se 75

of

the

spectra

the

RESEARCH

and

straight-chain

Infrared

than

for

1%6),

fairly

Se.

BIOPHYSICAL

requirements

(Trutter,

weight

y bound

AND

Se and 270:1

specific compared

in of

a recovery

the

of

radioactivity

characterized free

fatty activity’, with

fraction acid.

Based the

a theoretical

second

Vol.

11, No.

BIOCHEMICAL

1, 1963

ratio

of

7:1

would

imply

R-<-Se-R

that

molecular Many clusive

,O

for

only is

other

physical

results In

or

general,

not

al 1 of

ing

ester

which

free-acid is

also

may

strongly Since

occur

in

I abel

it

lower

larger

of

The

p rove,

in

that

the

of

the

al ways

seleno-esters, 0 as R-CiSe-R.

present one

may

crude-wax

A larger

before

one

and share

fraction,

Se will

least

preparation

count.

one

that

at

which

is

reof

the

of

conMuch

constituents.

further

some

1962),

expect

separated

treatshow

of

is

lack-

various

carbonyl

crystallizable

needed

of

(Renson,

crude-wax the

fractions

to

al ready

the

those

fractions,

according

Se

constant

than be

incon-

produces

because

mixtures,

from

residual

may

with

nevertheless

ester

We have

nearly

the

mostly

in

which, acid,

complex

weight

be

make

of

may

significantly

of

Stanleva

part of

of the

clear

that

wax.

a chemical

worthy

part

it

leaf

appearance

adaptation differ

to

material

is

seems to

or

which

lacking

Since,

some

material

Stanleva

Se

mere

paration,

least

times

presented

with

The

This

significant

studies

can

structure.

that

proves

of

data

associated

at

made

procedure

always

displaced

groups.

molecular

amounts

made

the

any

organic

themselves

remains

, however,

siderably

be

five

is

5.8%.

of

waxy

crystallized

selenium

been

Fractions

free at

functional

have

in

label

any

that

high-molecular-weight

beeswax,

label.

spectra.

location

various

of

, the

of

are

to

contained

tests

that

absorption

waxes

in

loss

I abel

suspect

COMMUNICATIONS

equivalent

molecules

significant

devoid

the

wax

chemical

carbonyl

carbonyl

this

40

and

have

be

in

RESEARCH

high.

is

acid

should

a molecular-weight

complete it

and

ments,

very

BIOPHYSICAL

of one

weight

AND

It of

wax is

suggests,

the

wax

Se absorbed reporting

at

this

indicated, that

and of

sulfur.

12

the

of

does

Se

not

is

intimately

conclusively

molecules. the time.

then

amount but

through

molecules,

from

a fair

an

roots, If,

as

interesting

metabolism

in

a leaf-wax

seems

likely,

preSe

biochemical of

Se

in

these

plants

Vol. 11, No. 1, 1963

BIOCHEMICAL

AND BIOPHYSICAL

Acknowl

The acid

authors

chromatography The

by this

wish

the

also

National

G.

and

assistance

not

his

gratefully

Institutes

could

have

edqements

acknowledge

method

authors

work

to

RESEARCH COMMUNICATIONS

J.

Miller’s

suggestion

in

acknowledge

the

of

Health

through

been

carried

on.

carrying

it

financial

Grant

of

the

Lis

sllicic

out. assistance

EF-229(Cl),

furnished

without

which

References Beath,

0.

(1947)

A.,

2.

Feigl,

3.

Hirsch,

4.

Klein,

5.

Lis,

6.

Renson,

M.

7.

Shrift,

A.,

8.

Trelease, Printers,

9.

and

Eppson,

H.

F.,

Univ.

of

Wyo.

Ag.

Exp.

Sta.

Bull.

278

No.

. F.,

Anal.

J.

and

A. E.

Trutter, New York,

Acta.

Ahrens, J.

K.,

W.,

Chim.

J.

Dragnet, Bot.

and

&,

Beath, N. Y.,

Wool Wax, (1956).

501

Jr.,

R.,

Bull. 550

Bi’ol

Chemists

Okey,

. Chem. 24,

Anal. Sot.

311 (1958).

a,

363

(1947).

Biochem.

2,

Belg.,

z,

Chim.

100

260

(1961). (1962).

(1958). Selenium, Out of

0. A., (1949). 368

(1961). J.

Agr.

C.,

Rev.

S. F. and New York, E. V., N. Y.,

H.,

ASSOC. Off.

Tinoco, and

E.

24,

pp.

13

Illus.

292

pp.

lllus.,

Champlain

print. Interscience

Pub1

ishers,

Inc.,