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Md.Mohiuddin
MBI/3511/09
u he accurate determination of most of the amino
acids of proteins is developed by the advent of
chromatographic methods.

But:-Analysis for tryptophan has remained a special

problem.
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u pectrophotometric measurement of tryptophan
Practically used for the soluble protein
Advantage:-Avoid the problem of hydrolysis.
u Magnetic circular dichroic absorbance at 293 nm
Advantage:-Gives accurate estimates of tryptophan in
several proteins,
Disadvantage:-his method requires highly specialized
equipment.
 But why we need determination of the tryptophan
content of protein by ion exchange
chromatography of alkaline hydrolysis.

u Because:-
All the above procedure is inapplicable when
the protein are insoluble.
Like:- Case in the analysis of food.
ame problem to the methods design to give a
colored derivative of tryptophan in the intact protein
 ome method of hydrolysis is to be
used,
he most convenient approach might be to modify
acid hydrolysis.
Chemical Hydrolysis
u Matsubara and asaki:-
he addition of mercaptans to 6 N HCl in the absence of
oxygen improves the recovery of tryptophan when
carbohydrate is absent.
u Liu and Chang:-
Made the valuable observation that p-toluenesulfonic acid
containing 3-(2-aminoethyl) indole is much preferable to HCl
in terms of the stability of tryptophan.
 ŒGiving about 90% recovery of tryptophan in a 22- 22-hour
hydrolysate of a purified protein; If the carbohydrate
content of the sample is appreciable, the recovery is
decreased.
Enzyme hydrolysis:-
ŒIt is an attractive alternative
ŒBut as yet it has not proved that capable of giving complete
hydrolysis in all instance.
 Conclusion from the above both
chemical and enzymatic hydrolysis
u Alkaline hydrolysis is the best way of obtaining a
quantitative recovery. Because complete hydrolysis
is desired for chromatographic determination of
tryptophan.
 EXPERIMENAL PROCEDURE
he following experiments were designed to determine the recovery
of tryptophan under various conditions of hydrolysis in NaOH.
NaOH.
Materials--
Materials

u L-ryptophan u Bovine pancreatic
u L-ryptophyl-L-leucine DNase A
u L-isoleucyl-L-tryptophan u Recrystallized (6 times)
egg white lysozyme
u Lysinoalanine
u Pepsin
u Bovine serum albumin
u pepsinogen
u Half-cystinyl human serum
albumin u Bovine thyrotropin
u Recrystallized bovine cr-
chymotrypsinogen
(CD541),
 Materials
u bovine cr-chymotrypsin(CD1 8GA),
u bovine trypsin (Rl 6257), and bovine pancreatic
u RNase A (RAF OAB)
u Defatted maize meals
u Hecker·s commercial unbleached wheat flour
u Unmodified potato starch
u Connaught hydrolyzed potato starch for gel
electrophoresis
 Hydrolysis of Proteins
u Quantitative recoveries of tryptophan are obtained when proteins (1
to 5 mg) are hydrolyzed at 110µ or 135µ in 0.6 ml of 4.2 N NaOH
containing 25 mg of starch.
Hydrolysis is performed in :-
Polypropylene liners sealed inside glass tubes evacuated to below 50
micro meter of mercury.
u Pyrex tubes in order to avoid silicate formation during alkaline
hydrolysis.
u he use of NaOH instead of Ba(OH)2 to avoid loss of tryptophan by
adsorption on BaO,4 or BaC03;
 Because:--
Because:

uUse of Ba(OH)2 requires the precipitation of

Ba++ by o4= or CO3=, with the problem of
adsorption of tryptophan to the precipitate
And:-
u Use of NaOH; this base can be neutralized and the
resulting NaCl does not interfere with the
chromatography
Use of Partially Hydrolyzed Potato starch
u Potato starch was partially hydrolyzed in acid in
order to increase its solubility in the hydrolysis
medium for protein.
u he inclusion of starch as the most effective
antioxidant tested;
 Chromafography--
Chromafography
u Chromatography with a buffer which separates tryptophan from
N-(DL-2-amino-2- carboxyethyl)-L-lysine, which can be formed in
significant quantities during alkaline hydrolysis.
u A 1-ml sample of the neutralized hydrolysate at pH 4.25 was added
to a column (0.9 x 8 cm or 0.9 X 12 cm) of Beckman PA-35 cation
exchange resin.
u he column were operated at a buffer flow rate of 50 ml per hour
and at 52 Degree.
u he eluent was sodium citrate at pH 5.4, 0.21 N in Na+ (A 3 to 5
dilution of the pH 5.3 buffer for the short column.
Note:- he length of the column required for adequate separation
dependon the molar excesses of the acidic and neutral amino acids
present relative to tryptophan
 REUL
pH of Buffer for ample Addition-
Addition-
u It is customary with an amino acid analyzer to use a pH-2.2 to
dilute the sample for analysis.
u BU:- ryptophan may be labile in acid solution, the recovery of the
amino acid was checked after different periods of standing in the
pH 2.2 buffer.
u At room temperature, the loss of tryptophan is significant (7% in 20
hours).
u ince the ion exchange column used for tryptophan analysis is
buffered at pH 5.4,
u herefore, the diluent for the hydrolysate for tryptophan analysis is
a buffer at pH 4.25 in which the loss of tryptophan overnight is
negligible (less than 1%.)
Fig--2
Fig
Effectiveness of tarch in Protecting ryptophan
under Alkaline Hydrolytic Conditions
Recoveries of ryptophan from Purified Proteins
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 DICUION
u teps in this procedure are documented in terms of
chemical parameters which contribute to the
quantitativeness of the recovery of tryptophan
u Effective protective action of starch is somewhat
unexpected and cannot be explained in detail.
u he protective effect of starch is not sufficient,
however, to permit the hydrolysis to be conducted
without the removal of most of the oxygen by
evacuation.
u If the tubes are not brought down to below 50 pm,
the recovery of tryptophan falls off
u Acid hydrolysis and alkaline hydrolysis will both have
uses in the determination of tryptophan.
u he method is advantageous in that tryptophan is
measured in the same hydrolysate used for the
determination of the other amino acids.
u he ability to obtain quantitative recovery of
tryptophan by alkaline hydrolysis will be useful in
experiments on peptide synthesis when it is
desired to determine the yield in the
incorporation with maximum precision.