Effect of different alkalies, temperature, and hydrolysis times on tryptophan determination of pure proteins and of foods

ANALYTICAL

BIOCHEMISTRY

109, 192- 197 (1980)

Effect of Different Alkalies, Temperature, and Hydrolysis Times on Tryptophan Determination of Pure Proteins and of Foods BERNARDO Ditisicin

de Estudios

de Posgrudo.

Fwuliud

LUCAS

AND ANGELA

de Quimicu.

I/NAM,

Received

May

Ciudad

SOTELO Unirc>rsitariu,

Mc;sico

20. D.F.,

Mcxko

19. 1980

A comparative study was carried out in order to determine which of the most commonly used alkalies for protein hydrolysis in tryptophan determination gave the best results. Hydrolyses were performed with 2.5 and 4N Ba (OH),, 4 and 1ON NaOH. 5 N NaOH containing 5% SnCI,, and with 4~ LiOH, not previously reported for use. The effect of temperature and hydrolysis time on the measured tryptophan content was also determined. Based on results obtained with lysozyme and with seven high protein preparations 4~ LiOH gave the best results. A temperature of 145°C was selected as the most convenient temperature since maximum tryptophan values were obtained with 4-8 h. The hydrolysis time required was inversely related to the protein content of the preparation. Lysozyme, casein. bovine plasma protein, and dehydrated whole egg gave maximum tryptophan content after 4 h hydrolysis while skimmed milk powder, rice flour, wheat flour. and wild legume flour required 8 h hydrolysis.

After tryptophan was isolated following enzymatic hydrolysis of casein, (1) many chemical and microbiological methods have been proposed for the determination of this essential amino acid in pure proteins or in foods. Problems in tryptophan determination are not only related to the quantitative measurement of the amino acid (2- 10) but also related to the difficulty of obtaining complete hydrolysis of the protein without some loss of the amino acid. Most of the chemical methods for tryptophan determination are based on the original method of Spies and Chambers (11) using Ehrlich’s reagent: p-dimethylaminobenzaldehyde (PDBA)’ in acid medium to produce a blue color following oxidation with NaNO,. This reaction with tryptophan occurs without hydrolysis of the protein; however. several authors reported a partial hydrolysis with enzymes which gave more quantitative results (12- 14). Other authors have reported ’ Abbreviation aldehyde; Trp.

used: PDBA. tryptophan.

0003-26971801170

19206$02.00/O

Copyright L 1980 hy Acadcmc be\\. All nghta of reprrrduct~m in any fcvn

p-dimethylaminobenz-

192 Inc. rcserrrd

that the PDBA reaction gave different results when it is carried out with tryptophan containing peptides than when it is done with free tryptophan (2). Although the PDBA method has been proposed for tryptophan analysis in foods, some plant materials contain compounds which interfere with the reaction of tryptophan. The same difficulties are found with the original and modified Opienzka-Blauth methods (15- 17). Microbiological methods of tryptophan determination will not be discussed here. although prior protein hydrolysis is also required in order to obtain good results ( 18,19). The best way to assure that tryptophan is being measured is by ion-exchange chromatography in an amino acid analyzer. Therefore, it is very important to be sure that there is a complete hydrolysis of the protein since only free tryptophan will be determined. Based on the previous considerations, the present work was planned with the following two objectives: (a) to perform the alka-

TRYPTOPHAN TABLE TRYPTOPHAN

DETERMINATION WITH

DIFFERENT

1 AFTER

HYDROLYSIS

ALKALIES Hydrolysis

Condltmn,

Alkah

I lo”Ci?O h g Trpi 16 g N”

16”rra h g Trpi 16 g 6”

Lysoryme Lysorymr Lysoryme

4 N NaOH IO Y NaOH 5 Y NaOH + 5’7

4 54 4.x: 6 38

4 Y4 4.31 5 w

Lysozyme Lysoryme Lysozyme Skimmed-mdk powder Skimmed-milk powder Skimmed-mdk powder

2.5 N BatOH), 4 N Ba(OHll 4 ~1 LiOH

: x0 3 34 $90

IY6 2.5: 6.60

4 N NaOH

1.10

0 9

IO N NaOH

I.15

I 06

I 46

I.16

2.5 u Ba(OHl,

0.16

0 20

4 N BatOH),

0.12

0 42

Sample

snrl,

Skimmed-mdk powder Skimmed-mdk powder Skimmed-milk powder I’ i\verage

5 N NaOH SKI,

4 N LIOH of duplicate

+ 5‘7

I.21

I 20

and 4 N LiOH. Hydrolysis was performed at 145°C for 2 to 12 h. This part of the study was designed to select the optimum time of hydrolysis. Eight samples of pure proteins or foods were hydrolyzed with 4 N LiOH at 145°C for 2. 4, 6. 8, and 12 h, and the free tryptophan was determined in an amino acid analyzer. The purpose of this part of the study was to determine the effect of the protein concentration in the sample on the hydrolysis time required. The samples used were: (a) Egg white lysozyme (Sigma Co. St. Louis, MO.), (b) Casein (General Biochemicals, Chagrin Falls), (c) Skimmed milk powder (Conasupo, MexTABLE ALKALINE LrhlL)

&ND

AT 145

i-

VAR’~.ING

run\

line hydrolysis of pure proteins, and food stuffs with some of the commonly used alkalies and also with LiOH, not previously reported in the literature; and (b) to study how tryptophan recovery is affected when the hydrolysis is performed at different temperatures or periods of time. MATERIAL

193

DETERMINATION

2

HYDROLYSIS

OF- A PURE

01.

(SKIMMED-MILK

1°C

A

FOOD

USING

Two

DI~FFRFNI

~-IN (LEG POWDER)

AI

K~LILS

AND

L~NCXHSOFTIMF

Hydrolysis with 5 NaOH containing 5c; SnCl, Time (h)

Samples Lysozyme

AND METHODS

Comparison of the effectiveness of tryptophan determination following alkaline hydrolysis was determined with egg white lysozyme, a pure protein with known tryptophan content and with dehydrated skimmed milk. The alkaline solutions used for hydrolysis were: 2.5 N Ba(OH),. 4~ 4N NaOH, 10~ NaOH, 5N Ba(OHh, NaOH containing 5% SnCl,, and 4 N LiOH. The temperature and times used were: 110°C for 20 h and 145°C for 4 h. The short time and high temperature have been used by other authors in acid hydrolysis (20). Based on the results (Table I), two alkaline solutions were selected for additional investigation: 5 N NaOH 5% SnC1,

PKOI

Skimmed-milk Skimmed-milk Skimmed-milk Skimmed-milk

powder powder powder powder Hydrolysis

with

Lysozyme Lysozyme Lysozymr Lysozyme Skimmed-milk Skimmed-milk Skimmed-milk Skimmed-milk Skimmed-milk Skimmed-milk ” Average

powder powder powder powder powder powder of duplicate

runs

g Trp!lh

2.0 3.5 5.0 12.0

4.91 5.54 5.56 5.56

3.5 5.0 7.5 12.0

1.09 1.26 1.15 I.02

4

N

LiOH

3.0 3.5 5.0 I?.0

5.73 6.73 6.60 6.27

2.0 3.0 3.5 h.0 8.0 11.0

1.05 1.14 1.19 1.32 1.35 1.31

N

g

N”

194

LUCAS

AND

ice, D.F.), (d) Bovine blood plasma protein (Escuela de Ciencias Biologicas, I.P.N. Mexico, D.F.), (e) Dehydrated and defatted whole egg (prepared in the laboratory), (f) and (g) Wheat and rice flour (commercial products obtained in a market, Mexico, D.F.), (h) Mucuncr argyrophylla: wild legume seeds collected in Veracruz State, Mexico. Hydrolysis procrduw . The dry sample was placed into a 150 x 25mm culture tube fitted with a screw cap with Teflon liner (Pyrex brand, Curtin serial 9826). These tubes have been used successfully by us for acid hydrolysis. The amount of sample used was equivalent to 100 mg of protein, previously determined by micro Kjeldahl

SOTELO

method: the amount of alkali used was: 1 ml/ 25 mg of sample. Air was removed from the tube by nitrogen flushing: the sample was subsequently frozen in acetone-dry ice. The tube was tightly closed and the digestion was carried out in a Tecator digestor Model ab 20140 (Tecator Co. Boulder, Colo.) at the temperature and time desired. At the end of the hydrolysis time, the hydrolisate was neutralized with orthophosphoric acid (85%) using a pH meter. With Ba(OH)2. the BaZ+ was precipitate as barium phosphate (21) and the sample filtered. The filtrate was brought to a known volume and an aliquot was diluted with 0.2 M phosphate, pH 7 (22). A IOO- to I50-

NEUTRAL AND ACID AMINO ACIDS

LYS IS

TYR

PHE

NH3 TRYPTOPHAN 7 ARG

iI

FIG. I. Chromatographic separation of tryptophan from a standard column (250 x 5 mm) of Technicon C-3 resin was developed at a buffer at 55°C with 1.036 M (in Na’) sodium acetate buffer, pH 5.5.

mixture of amino acids. A flow rate of 37.2 ml per hour

TRYPTOPHAN

4 Hydrolysis

FIG. 2. Zero time value tained at different hydrolysis

6 Time

PROTEIN

CONTENT

AND

FOR MAXIMUV

Samples”

Protein content (%i)

Lysozyme Casein Protein isolated from bovine blood plasma Dehydrated whole egg Skimmed-milk powder Wheat flour Rice flour Mucuna argyrophyla seed (wdd

legume)

” Average of duplicate runs. ” From Fig. 3. ’ From: Amino Acid Content FAO. Rome. ” Actual tryptophan (IO).

‘b maximum value”

the experimental

ob-

3

HYDROLYSIS YILLII

TIMF

IN PLRF

PR~FLINS

Hydrolysis time (h)

Zero time value

Trp destroyed (3)

6.73 1.31

3.5 4.0

6.90 I.36

4.5 3.7

98.0 67.5 32.3 11.2 9. I

I .68 .s9 .35 .I4

4.0 6.0 8.0 8 .O

.50

x 0

1.76 I .68 1.40 I .20 1.57

4.5 5.4 3.6 5.0 4.5

14.7

1.49

x.0

I .60

6.8

and Biological

:AND

FOODS

OF TR~P~OPHAN

88.0 91.2

of Foods

values

ing temp. 55 -C O._S”C, flow rate: 0.62 ml/ min, and running time: 60 min. Prior to use, the resin was washed with 0.2 N NaOH and then with 0.05 M (Na+) sodium acetate buffer. pH 3.90. The elution buffer was 1.036 M (Na+) sodium acetate, pH 5.50. The amino acids standard (Sigma

TABLE ot

i ( hours)

of tryptophan calculated by extrapolating times in 4 N LiOH at 145’C.

~1 aliquot was injected on to a Technicon Amino Acid analyzer Model NC-2Pequipped with a single column (Technicon International, Geneva). The analyzer conditions were the following: Column size: 286 x 5 mm, resin bed height: 250 t 5 mm, resin type: C-3, operat-

RLLATIONSHIP

195

DETERMINATION

Data on Proteins

( 1970).

Nutritional

Reference (g Trp,‘l6

data’ g N)

7.09” I .O-2.74

1.01-1.80 1.18-1.60 l.Ol-I.?0

0.86-l

Studies

.YO

No. 24.

196

LUCAS

AND

temperature of 145°C was chosen in order to minimize the time of hydrolysis since no difference was found when compared to the 20-h hydrolysis at 110°C (P > 0.05). The data of Table 2 show that maximum tryptophan yields for lysozyme were obtained within 3.5 h. However, in the skimmed milk powder, a longer time was required to achieve maximum tryptophan yields. Better results were obtained by hydrolysis with 4 N LiOH than with 5 N NaOH containing 5% SnCI, (P < 0.05). The procedure proposed by Roach and Gehrke for acid hydrolysis (20) was used to correct for tryptophan destruction during hydrolysis as shown in Fig. 2. The results obtained when samples of pure proteins, protein concentrates and foods with low protein content were hydrolyzed for varying lengths of time using LiOH at 145°C are shown in Table 3 and Fig. 3. The time required to obtain the maximum value of tryptophan was in-

Chemical Co.) and the sample were diluted with 0.2 M phosphate buffer, pff 7.0 Ninhydrin (Sigma Chemical Co.) was prepared according to the Technicon manual (23). RESULTS

SOTELO

AND DISCUSSION

The aminogram of the standard amino acids mixture is shown in Fig. 1. Tryptophan was eluted at 44 min. The results obtained using the different alkali solutions are shown in Table I. The best results for tryptophan content of lysozyme and skimmed milk powder were with 5 N NaOH containing 5% SnC&, which has been used successfully by Lugg (24), and with 4 N LiOH. On the other hand, 2.5 and 4 N Ba(OH), gave the poorest results, under the same conditions of time and temperature. Based on these results, the hydrolysis was repeated with 5 N NaOH containing 5% SnCI, and with 4 N LiOH at 145°C for different periods of time. A

Tr A

PROTEIN

CONCENTRATION

9-35

RF =AICE FLOUR WF=WHEAT FLOUR SMP .SKIYHED MILK WL’WILD LEGUME

/16gN) 3

X

PROTEIN CONCENTRATION

64-91

%

L s LYSOZYME BP = BOVINE PLASMA DWE. DRIED WHOLE EGG C ‘CASEIN

POWDER 70 /+----wL 60

HYDROLYSIS

FIG. 3. Effect of hydrolysis time on measured hydrolysis conditions: 4 N LiOH at 145°C.

TIME

tryptophan

(HOURS)

content

of different

types

of samples.

TRYPTOPHAN

DETERMINATION

versely proportional to the protein content (Table 3). Extrapolation to zero time indicates that there was about 5% destruction of tryptophan with 4 to 8 h of hydrolysis. Our results indicate that under our conditions: (a) 4 N LiOH is the best alkali for protein hydrolysis; (b) the temperature of 145°C used in acid hydrolysis is convenient also in alkaline hydrolysis since hydrolysis time is considerably reduced and only 5% of the original tryptophan is destroyed; (c) under the conditions described here, the hydrolysis time for maximum tryptophan yield for pure or isolated proteins is 4 h and 8 h for the other foods. ACKNOWLEDGMENTS This research was supported by the Organization of American States. The authors thank to Dr. A. Parra and Professor J. R. Whitaker for helpful criticism. The secretarial work was performed by Mrs. Luz Ma. Luna.

REFERENCES 1. Greenstein, J. P.. and Winitz, M. (1961) Chemistry for the Amino Acids Vol. 2. pp. 23162324, Wiley, New York. 2. Miller. E. L. (1967) J. Sci. A@. 18, 381-386. 3. Westgarth, D. R., and Williams. A. P. (1974) J. Sci. Agric. 57 I-575. 4. Sullivan, B. X.. and Hess. W. C. (1944) J. Biol. Chrm. 155,441-446.

197

5. Knox, R.. Kohler. G. 0.. Palter, R.. and Walker. H. G. (1970) Anal. Biochem. 36. 136-143. 6. Spies, J. R.. and Chambers. D. C. (1949) Ann/. Chem. 21, 1249- 1266. 7. Hugli. T. E.. and Moore, S. t 1972) J. Bid. Chrm. 247, 2828-2834. 18, 406-413. 8. Robel, E. J. (1967) Am/. Bivchem. 9. Matheson. N. A. (1974) Rrir. J. Nurr. 31, 393400. 10. Spies. J. R. (1967) Anuf. Chem. 39, 1412- 1416. 11. Spies. J. R., and Chambers. D. C. (1948) Anal. Cht~m. 20, 30-39. I?. Lombard. J. H.. and Lange, D. J. (1965) And. Bioc~hrm. 10, 260-265. 13. Spies. J. R. (1968) .I. Agr. F~wd Chrnr. 16, Sl4516. 14. Perreird. P. G. t 1972) Arc,/z. Lafimwruc~r. h’trrr. 22, 283-290. IS. Opienzka-Blauth. J., Charezinski. M.. and Berbet, H. (1963) And. Biochem. 6, 69. 16. Villegas. E.. and Mertz, E. T. (1971) Rrs. Bull. No. 2 Cimmyt, Mexico. 17. Concon. J. M. (1975)AnuI. Biochem. 67,206-219. 18. Green, R. D.. and Black. A. (1944) J. Bid. (‘hem. 155, 1-8. 19. Henderson. L. M.. and Snell, E. E. (1948) J. Bid. Chrm. 172, 15-29. 20. Roach, D.. and Gehrke. C. W. t 1970) J. Chromtogr. 52, 393-404. J. t 1965) Analisis Cualitativo y Quimica 21. Norman, Inorg&rica. pp. 359-360, Continental. Mexico. 22. Heppel, L. (19551 in Methods in Enzymology (Colowick. S. P., and Kaplan, N. 0.. eds.). Vol. 1, pp. 143, Academic Press, New York. 23. Technical Report No. 9 (1974) Technicon International Division, Geneva. 24. Lugg. J. W. E. (1938) Bir~henz. J. 32, 775-783.