Determination of the Tryptophan Content of Proteins By

8/8/2019 Determination of the Tryptophan Content of Proteins By

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Determination of theDetermination of theTryptophan Content ofTryptophan Content of

Proteins by IonProteins by Ion

Exchange ChromatographyExchange Chromatographyof Alkalineof Alkaline HydrolysatesHydrolysates**

Md.Mohiuddin

MBI/3511/09


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y The 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 specialproblem.


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Various approaches for the determination ofVarious approaches for the determination of

this amino acid are studied.this amino acid are studied.

y Spectrophotometric measurement of tryptophan

Practically used for the soluble protein

Advantage:-Avoid the problem of hydrolysis.y Magnetic circular dichroic absorbance at 293 nm

Advantage:-Gives accurate estimates of tryptophan inseveral proteins,

Disadvantage:-This method requires highly specialized

equipment.


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But why we need determination of the tryptophanBut why we need determination of the tryptophan

content of protein by ion exchangecontent of protein by ion exchangechromatography of alkaline hydrolysis.chromatography of alkaline hydrolysis.

y Because:-

All the above procedure is inapplicable whenthe protein are insoluble.

Like:- Case in the analysis of food.

Same problem to the methods design to give acolored derivative of tryptophan in the intact protein


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Some method of hydrolysis is to beSome method of hydrolysis is to be

used,used,The most convenient approach might be to modifyacid hydrolysis.

Chemical Hydrolysis

y Matsubara and Sasaki:-The addition of mercaptans to 6 N HCl in the absence ofoxygen improves the recovery of tryptophan whencarbohydrate is absent.

y Liu and Chang:-Made the valuable observation that p-toluenesulfonic acidcontaining 3-(2-aminoethyl) indole is much preferable to HClin terms of the stability of tryptophan.


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Giving about 90% recoveryGiving about 90% recovery of tryptophan in a 22of tryptophan in a 22--hourhour

hydrolysatehydrolysate of a purified protein;of a purified protein; If the carbohydrateIf the carbohydratecontent of the sample is appreciable,content of the sample is appreciable, the recovery isthe recovery is

decreased.decreased.

Enzyme hydrolysis:-It is an attractive alternative

But as yet it has not proved that capable of giving complete

hydrolysis in all instance.


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Conclusion from the above bothConclusion from the above both

chemical and enzymatic hydrolysischemical and enzymatic hydrolysisy Alkaline hydrolysis is the best way of obtaining a

quantitative recovery. Because complete hydrolysis

is desired for chromatographic determination oftryptophan.


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EXPERIMENTAL PROCEDUREEXPERIMENTAL PROCEDUREThe following experiments were designed to determine the recoveryThe following experiments were designed to determine the recovery

of tryptophan under various conditions of hydrolysis inof tryptophan under various conditions of hydrolysis in NaOHNaOH..

MaterialsMaterials--..

y L-Tryptophan

y

L-T

ryptophyl-L-leuciney L-isoleucyl-L-tryptophan

y Lysinoalanine

y Bovine serum albumin

y Half-cystinyl human serum

albumin

y Bovine pancreatic

DNase A

y Recrystallized (6 times)

egg white lysozyme

y Pepsin

y

pepsinogeny Bovine thyrotropin

y Recrystallized bovine cr-

chymotrypsinogen

(CD541),


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MaterialsMaterials

y bovine cr-chymotrypsin(CD1 8GA),

y bovine trypsin (TRl 6257), and bovine pancreatic

y

RNase A (RAF OAB)y Defatted maize meals

y Heckers commercial unbleached wheat flour

y Unmodified potato starch

y Connaught hydrolyzed potato starch for gel

electrophoresis


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Hydrolysis of ProteinsHydrolysis of Proteins

y Quantitative recoveries of tryptophan are obtained when proteins (1

to 5 mg) are hydrolyzed at 110 or 135 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.

y Pyrex tubes in order to avoid silicate formation during alkaline

hydrolysis.y The use of NaOH instead of Ba(OH)2 to avoid loss of tryptophan by

adsorption on BaSO,4 or BaC03;


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Because:Because:--

y Use of Ba(OH)2 requires the precipitation of

Ba++ by So4= or CO3=, with the problem of

adsorption of tryptophan to the precipitate

And:-

y Use of NaOH; this base can be neutralized and the

resulting NaCl does not interfere with the

chromatography


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Use of Partially Hydrolyzed Potato starchUse of Partially Hydrolyzed Potato starch

y Potato starch was partially hydrolyzed in acid in

order to increase its solubility in the hydrolysis

medium for protein.

y The inclusion of starch as the most effective

antioxidant tested;


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ChromafographyChromafography--y Chromatography with a buffer which separates tryptophan from

N-(DL-2-amino-2- carboxyethyl)-L-lysine, which can be formed insignificant quantities during alkaline hydrolysis.

y 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) ofBeckman PA-35 cation

exchange resin.

y The column were operated at a buffer flow rate of 50 ml per hour

and at 52 Degree.

y The 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:-The length of the column required for adequate separationdependon the molar excesses of the acidic and neutral amino acids

present relative to tryptophan


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RESULTRESULT

pH of Buffer for Sample AdditionpH of Buffer for Sample Addition--y It is customary with an amino acid analyzer to use a pH-2.2 to

dilute the sample for analysis.

y BUT:-Tryptophan 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.

y At room temperature, the loss of tryptophan is significant (7% in 20

hours).

y Since the ion exchange column used for tryptophan analysis is

buffered at pH 5.4,

y Therefore, 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%.)


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FigFig--22


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Effectiveness ofStarch in ProtectingTryptophanEffectiveness ofStarch in ProtectingTryptophan

under Alkaline Hydrolytic Conditionsunder Alkaline Hydrolytic Conditions


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Recoveries ofTryptophan from Purified ProteinsRecoveries ofTryptophan from Purified Proteins


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Stability of Tryptophan Residues in Proteins in AcidicStability of Tryptophan Residues in Proteins in Acidic

MediaMedia--


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DISCUSSIONDISCUSSIONy Steps in this procedure are documented in terms of

chemical parameters which contribute to thequantitativeness of the recovery of tryptophan

y Effective protective action of starch is somewhatunexpected and cannot be explained in detail.

y The protective effect of starch is not sufficient,however, to permit the hydrolysis to be conductedwithout the removal of most of the oxygen byevacuation.

y If the tubes are not brought down to below 50 pm,the recovery of tryptophan falls off

y Acid hydrolysis and alkaline hydrolysis will both haveuses in the determination of tryptophan.


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Determination of the Tryptophan Content of Proteins By