Determination of tryptophan in feeds

ANALYTICAL

BP.XHFXlNTRY

36,

Determination

136-143

i

1!)70)

of Tryptophan

in feeds

RANDY KNOX, G. 0. KOHLER, RHO.UA FALTER> AND I-I. G. W,41,KER.

Much of the literature available on amino acid content of feeds lacks accurate tryptophan values owing to difficulties of tryptophan analysis. Three representative tryptophan methods commonly employed were found by Miller (1) to be unsatisfactory for the routine analysis of feeds. These included a microbiological method (2) and two colorimetric methods (3, 4). The modified method of Spies (5) is useful for the determination of tryptophan in isolated proteins but has not proved satisfactory for use with feeds in g,eneral (6) because of interference by unknown factors in calorimetric determinati.on of t’ryptophan. Miller (I) developed a method of Ba (OH), hydrolysis followed by :L p-dimethylamino benzaldehyde (D&IAB) determination of tryptophan in feeds. A key feature of his method was prec.ipitation of barium from an acid medium prior to nddit,ion of DMAB. His method gave only 80% recovery of added tryptophan for maiztl and 84% recovery for wheat. Slum,p and Schreuder (7) recently published a method eliminating Miller’s precipitation step. They used a low pH b,uffer (pH 3.25) tIo avoid precipitat’ion of barium while eluting hgdrolyzate from a Sephadex G-25 column. Although ~*ecoveryof added tryptopharl was high (94 to IOO%), 80 min was required t’o resolve the trypt,ophan peak, limiting the number of samples which can bc run daily. The present paper reports improvements on the: ion exchangeprocedural for tryptophan analysis developed in our laboratory (8) prior to the appearance of the Slump and Schreuder report’ (7). A very recent8report’ by Tkachuk and Irvine indicates that they have worked out a method similar to ours (9). They do not, however, report any comparat’ive results between the ion exchange and other tryptophan methods. In both procedures samples are hydrolyzed in Ba(OH)2 solut,ion in evacuat’ed,sealed tubes. Barium is precipitat’ed from an acid medium and tryptophan determined on t,he basic column of an amino acid analyzer. High recoveries 136

DETERMINATION

OF TRYPTOPHAN

137

of added tryptophan (approximately 94%) are obtained. Sincetryptophan is resolved within 25 min, it is possible to analyze a relatively large number of samples daily by using two columns, one for running sample while t’he other is being regenerated and loa.ded. MATERIALS

Lysozyme (egg white), 3~ crysta.llized; hemoglobin (human), 100% pure by electrophoresis; nn-tryptophan 13.68%AT (E,,,,, 217 rnp 33,900, 288 my 5,350, and 287 rnp 4,490) were purchased from Mann Research Laboratories, New York-l Other protein samples were supplied through the courtesy of Joseph 12. Spies (5), Dairy Products Laboratory, Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Depart,ment of Agriculture, Washington, D. ‘C. Feed samples were obtained as follows: Montana Hard Red Spring (FIRS) wheat from Fisher Flouring Mills; alfalfa from Dickson Dehydrator Co. at Dixon, California; bran from HRS whe,at from International Milling at Minneapolis ; and s&lower meal from J. C. Boswell Co. of Corcoran, California. METHOD

A ball-milled sample containing approximately 6 mg nitrogen (according to previous elemental ana’lysisj is added to a constricted-neck tube (25 x 200 mm Pyrex test tube or 30 ml Pyrex micro-Kjeldahl flask) containing 5.0 gm Ba (OH),*SH,O. The sample js washed into the tube with 4.0 ml water. Four s,uchtubes filkd with the sample are attached to outlet,s of a high-vacuum system. The contents are frozen by immersing the tubes in liquid N,, and tubes are evacuated to at least 20~ pressure while being kept cool. The tubes arc isolat,ed from the vacuum pumping system and while still under vacuum are cautiously warmed to room temperature. They are refrozen and evacuated to 15~~ isolated, and then warmed, frozen, and evacuated again. When the pressure is less than 10~ the constrictions are sealed with a gas oxygen torch. The samples are warmed to room temperature and placed .in a 110 & 1” oven for 1616 hr. At the. conclusion of this period they are cooled .te rodm temperature, opened, and rinsed into 100 ml bottles. ~Eaah,sample- is titrated to pH 2 with 6 iV HCI while being .stirred lmagnetically. Stirring is continued as 25 ml of. Na2S04 solution (91 gm anhydrous. NasSOr/liter) is added tJo precipitate barium. The resuIti@ *Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.

138

KNOX ET AL.

suspension is transferred qusntitat.ively to a 50 ml volumetric flask and diluted to volume with H,O. The well mixed suspension.is centrifuged for 30 min at 14,600g in a 250 ml polypropylene centrifuge bottle. 4 ml of clear supernatant is added to a basic column of a Beckman Spinco model 120 amino acid analyzer. This high-pressure column is 0.9 X 23 cm packed to a height of 7 cm with Beckman PA-35 resin. Column temperature is maintained at 65”. Two aliquots of pH 3.25 buffer (10) (0.5 and 1.0 ml) driven by air pressure (20 psi) are used to rinse the sample into the resin After a further rinse with 0.5 ml of pH 5.00 buffer described below, the sample is eluted from the column with additional pH 5.00 buffer. Flow rates are TO ml/hr for buffer and 35 ml/hr for ninhydrin. Columns are regenerated with 0.2 N NaOH driven by 20 psi of air pressure, The pH 5.00 buffer is prepared by dissolving 501.0 gm sodium citrate dihydrate in water and adding to a container c.alihrated to contain 20.0 liters. An aqueous solution of 27.0 gm of disodium ethylenediaminetetraacetate (EDTA) is added fo8110wed by 130 ml concentrated HCl, 2.0 ml octanoic acid, 16.0 ml thiodiglycol and 30.0 ml detergent’ solution (200 gm + 400 ml H,O), respectively. The solution is diluted to 20 liters with deionized distilled water. Concentrated HCI or 50% NaOH is used to correct the pH to 5 4: 0.02. The resulting solut’ion is 0.255 N in sodium ion. Result’s of the analysis are expressedas gm tryptophan/l6 gm N. A solution of r&-tryptophan in pH 2.2 buffer (10) was used as a standard. The apparent tryptophan value in feed stuffs is multiplied by the correction factor, 1.069 (see Discussion). RESULTS

AND

DISCUSSION

Changing to a vacuum-,sealed hydrolysis technique eliminated much of the problem of erratic recoveries of added tryptophan encountered in previous work using a refhrx method (8’). Precipitation of barium from an acid medium a,s suggested by Miller (1) further increased the apparent tryptophan content of Montana Hard Red wheat by approximately 6%. The protective reagents (histidine and basic lead acetate) used by Spies (5) did not noticeably alter the tryptophan content thus obtained. A hydrolysis time of 16 to 18 hr (overnight) was chosen for convenience even though hydrolysis is essentially complete in 4 hr (see Fig. 1). Although the conventional pH 5.2$ buffer (8) fails to resolve tryptophan on a PA-35 resin basic column, a pH 5.00 buffer of lower sodium concentration gives the desired separation (see Fig. 2). Under these conditions, tryptophan is clearly separated from the natural protein amino acids and other ninhydrin reacting hydrolysis

DETERMINATION

OF

139

TRYPTOPHAN

.4

FIG. 1. Duration

of Ba(OH)z

hydrolysis

for tryptophan

at llO”.

HEMOGLOBIN

HRS WHEAT

rRYPTOPliAN 4 ?

MINUTES

FIQ. 2. Sample chromatograms obtained on 7 X 0.9 cm Beckman PA-35 resin columns eluted with pH 5 buffer: (a) Composite of some neutral and acidic peaks. (b) Ornithine lysine peak. (c) An unidentified minor peak, possibly hydroxyIysine.

(1No cturl’ection factor applied. I1Average of (found/integer) X 100 is 93.3 S.E. & 1.24. c Compiled by Spies (5). The molecuIar weight of hemoglobin is based on sequence studies (19, 20). The correct integer value for canalbumin is uncertain (see text). d By method of Slump and Schreltder (7i, 7.iOcjc, t’ryptophan. c Not Mbfl’w.

DETERMINATION

OF

141

TRYPTOYHAK

this basis we agree with Spies (5) and Lewis et al. (14) on a nearest integer of 11 tryptophan residues. Recent, work, however, gives values of 15 and 16 tryptophan residues respectitlely for this protein (15, 16) + These higher values were obtained by an indirect’ spectrophotometric method (17) and not by direct determinat’ion so that some discrepancy between the two methods may be justified. The nearest integ,er (-7) found for tryptophan in a-chymotrypsin as well as that determined by Spies (5) and that calculated from the data of Slump and Schreuder (7) was lower than that (=S) calculated from the amino acid sequencedctermined by Hartley (18). This may reflect tryptophan dest’ruction as shown by Spies and Chambers (3). A value found/integer x 100 was determined for those proteins whose molecular weight was we11estabIished. The average va1u.efor t,his ratio was found to be 93.3. Recoveries of pure tryptophan and tryptophan added to samples of wheat, aIfalfa, safflower meal, and wheat, bran a’rc given in Table 2. Recoveries run from 90.2 to 95.7% and are lowest for bran. Both recoveries of free tryptophan and tryptophan added to feed sampIes are approximately the same. Further, t’hcy are approximately equal to the values calculated earlier for proteins (found/integer) >( 100, although this may b’e coincidental. It suggest’sthere is a relatively constant percent,ageof tryptophan destruction a.nd that a correction factor should be used with many samples to correct apparent values to onfls more closccly TABLE 2 Recovery of Trypt’ophan Trypt)ophan

added, pmoles

0

Sample

gm,‘lS gmN

Tryptophan HRS wheat Dehydrated alfalfa HRS wheat bran SafRower meal Starch (250 mg) Glucose (250 mg) Sucrose (250 mg)

1.29 2.11 2.05 1.27 -

a Standard parentheses.

93.9 i 4.9 (5)" 90.7 +_ 5.2 (Sj -

error of mean (95y0 confidence

level)

93 7 k 3.9 (51 95 o+20(5) !)I 5 + 5.1 (61 610 2 4 8.0 (3 1 95 7(21 95 1 -i- 4.5 (41 x2 6(3 i 76 5i2! with

number

90.5

f

4 5 (*a ---

-‘-of

replicates

in

1.42

JsNI)X

ET

AL.

representing the a’ctual amount, present. Such ti factor was c:zlculat.ed as the reciprocal of the average recovery determined from the data irk Table 2 for 1.25 pmoles added tryptophan for all samples except8glucose and sucrose.These were neglected becausethey seemedto be abnormally low. The recovery thus determined was 93.57,, standard error of mean 3.0 (95% confidence level), so that the correction factor is 1.06'9. Spies’ earlier work in this field (3) shows clearly that tryptophan in prot8einis destroyed even more rapidly than free tryptophan in solution. Hence, amounts of tryptophan found in feedstuffs by t’his method probably represent minimal values. Special correction factors may be needed for commodit,ies with a high free-sugar content. Samples of Iysoeyme, wheat, and safflower (Tables 1 and 2) were analyzed by the method of Slump and Schreuder (7) and gave values averaging 97% of the uncorrected values determined by the method reported in this paper. Analysis of a sorghum sample (hydrolyzed by our method) on Slump and Schreuder’s Sephadex G-25 column (7) gave results similar to those obtained on our PA 35 column, which st’rengthens confidence in the reliability of bot,h methods. The over-all agreement shown between our values, the values reported by Spies (5>, and the values determined by the method of Slump and Schreuder (7) indicates that any Noneof these three methods, within limitations mentioned previously, is probably suit’able for routine analysis of tryptophan. Although Slump and Schreuder (7) ,avoid precipit’ation of ‘barium, we find that solut,i,onsobtained by their procedure have enough turbidity to require centrifugation. Because of t’he shortened column time the presently reported method offers a useful alternative if the analysis of a large number of samples is ContempIated. REFERENCES 1. MILLER, E. L., J. Sci. Food Agr. 18, 381 (1967). 2. GREENE, R. D., AND BLACK, A. J., J. Biot. Chem. 155, 1 (1944). 3. SPIES, J. It., AND CHAMBERS, D. C., Anal. Chem. 21, 1249 (1949). 4. HORN, M. J,, AND JONES, D. B., J. Viol. Chem. 157, 153 (1945).

5. SPIES, J. R., Anal. Chem. 39, 1412 (1967) + 6. SPIES, J. R., J. Agr. Food Chem. 16, 514 (1968). ‘7. SLUMP, P., AND i%XXREUDER, H. A. W., A&. &Xhem. 27, 182 (1969). 8. KOHLER, G. O., AND PALTEX, R., Cereal Chem. 44, 512 (1967). 9. TJSACEUK, B., AND IRVINE, G. N., Cereal Chem. 46, 206 (1969). 10. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Anal. Chem. 36, 1185 (1958). 11. WARNER, R. C., AND WEBER, I., J. Am. Chem. Sot. 75, 5@34 (1953). 12. PHELPS, R. A., AND CANN, J- R., Arch. Biochem. and Biophgs. 61, 51 (1956). 13. ‘J~MASHEFF, S. N., AND TINOCO, I., Arch. Biochem. and Biophys. 66, 427 (1957).

DETERMINATION

OF TRYPTOPWAPI;

143

14. LEWIS, J. C., SNELL, N. S. HIRSCHMANN, D. J., AND FRAENICEL-C~NR.ST, H., J. Bid. Chem. 186, 23 (1950). 15. AZARI, P., AND BAUGH, R. F., Arch. Biochem. and Biophys. 118, 138 (MS’). 16. WILLL4MS, J., B&hem. J. 83, 355 (1962), 17. BENCZE, W. I+ AND SCHMEJ, K., Anal. Chem. 29, 1193 (1957). 18. HARTLEY, B. S., Nature 201, 1284 (1964). 19. HILL, R. J., .~ND KONIGSBERG, W,, J. Bid. Chem. 237, 3151 (1962). 30. KONIGSBERG, W., GOLDSTEIN, J., AND HILL, R. J,, J. Bid. Chem. 238, 20% (1963).