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REACTION

OF

NINHYDRIN

WITH

DIPEPTIDES

spectrophotometry ures of the purity

(11) for tyrosine of the preparations

peptides provided used in this study.

additional

meas-

Methods-The

ninhydrin

method of Moore and Stein (1,4) was used with

slight

modifications,

SnClz-ninhydrin

(1)

0.1

M

sodium

citrate

reagent buffer, depending contained on the nature pH 5.0, and 50 volume 0.112 M ninhydrin of the experiments. , per cent methyl 0.0035 The M SnC12 Cel-

,

losolve. 0.112 M

The components

ninhydrin,

0.0093

of the hydrindantin-ninhydrin

M

hydrindantin,

1M

sodium

reagent (4) were acetate buffer, pH

5.5, and 75 volume

100” was varied. merson photoelectric

per cent methyl

Cellosolve.

Routine

measurements

calorimeter.

The

were Beckman

made with

a Klett-Sum-

model DU

spectropho-

The reaction

period

at

tometer tures.

was used Theoretical

to determine color yield

,

the spectra

based

on

of the ninhydrin

reaction

the

extinction

coefficient

mix- for the

colored product

(12), was obtained with the reference amino acid, L-leucine.

The naphthoquinone

sulfonate

reaction,

described

in the legend for Fig. 2,

was a slight modification

of the method

of Troll

(13).

Differences

in Reaction

Results Rates of Diastereoisomers--It

was

initially

ob-

served that the relative

reaction

rates of the diastereoisomers4

L-Leu-L-Tyr

and n-Leu-L-Tyr the experimental sulted from the

with

ninhydrin

varied from 2- to 5-fold, depending

upon

conditions. reaction of

Evidence ninhydrin

that the with these

same colored product

re-

dipeptides

and

L-leucine

was provided by reaction mixtures,

the identical particularly

In order to obtain maximal

beyond

the

20

minute

period

The color yields from the

nature

of the spectra

(350 to 750 mh) of the

the

absorption

maxima

color

yields, the

reaction

which

is

routinely

used

dipeptides,

as

presented

at 403 and 570 rnp. period was extended for amino acids (1). in Fig. 1, increased

steadily,

whereas

the reference

compound,

L-leucine,

gave maximal

values

after the first

5 minutes.

L-Leu-L-Tyr

approached

the ‘Lmolar”

yield of

dilution

of the 1.2

Unexpectedly,

however,

a value of 2.

Furthermore,

ml. reaction

mixtures

to 2.0 ml. with

water

prior to heating

resulted

in a

substantial

decrease in the reaction

rate of D-Leu-L-Tyr.

Thus,

a wide

range of relative

color yields could be obtained from these diastereoisomers,

depending

upon the reaction

period and the composition

of the reaction

mixture. The following

experiment,s

ruled out the possibility

that

impurities

irl

the dipeptide

preparations

rates and the abnormally

might

account

for the

high

“molar”

color

yields:

differences in

reactiorl

(a) Theoretical

values

for try

tyrosine and nitrogen (11) and Kjeldahl

were nitrogen

obtained from determinations,

ultraviolet

spectrophotome-

respectively;

(b)

no

nin-

4 Enantiomorphs,

n-Leu-Gly

and

L-Leu-Gly,

react

at equal

rates.

,

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