Colorimetric screening assay for cystine plus cysteine in legume seed meals

ANALYTICAL

BIOCHEMISTRY

87, 641-647 (1978)

Calorimetric Screening Assay for Cystine Cysteine in Legume Seed Meals

plus

A calorimetric assay for cystine plus cysteine in pure proteins has been adapted to legume seed meals. The procedure involves incubation of legume seed meals for 1 hr at 38°C with sodium borohydride in 8 M urea, destruction of the sodium borohydride, and calorimetric determination of thiols produced with Ellman’s reagent. A comparison of values from this procedure and from performic acid oxidation of 33 legume seed meals is presented and shows good correlation for peas (Pisum sativum) and lentils (Lens culinaris), with somewhat equivocal results for beans (Phase&s vulgaris) and fava beans ( Viciu fuba).

The sulfur amino acids methionine and cystine are commonly recognized as the human-essential amino acids most limiting in legume seeds (1). Rapid assays for these amino acids could be useful in selecting for legume seeds of higher nutritional quality. An innovative gas-liquid chromatographic assay for methionine determination in legume seed meals, which measures methyl thiocyanate after treatment of seed meals with cyanogen bromide, has been developed (2). Since the only cystine assays we are aware of involve a rather time-consuming per-formic acid oxidation and subsequent amino acid analysis (3), hydrazinolysis followed by hydrogen sulfide determination (4), or kinetic assay (5), we sought to develop a more rapid assay for cystine plus cysteine. A method for pure proteins in which disulfides are reduced in an 8 M urea-sodium borohydride solution, followed by acid destruction of the borohydride and estimation of the thiols produced with Ellman’s reagent [S,S’ dithiobis(nitrobenzoic acid)] (6), was adapted to legume seed meals. The results of the calorimetric determination are compared with the results of previously published values obtained by pet-formic acid oxidation of the same legume seed meals. MATERIALS

AND METHODS

Legume seed meals that were the subject of a very valuable earlier study (7) on the use of total sulfur as a screening assay for sulfur amino acids were generously provided by Dr. S. L. MacKenzie. The nitrogen-bubbling apparatus was constructed from materials used for drip irrigation. The main nitrogen reservoir, constructed of polypropylene, was 40 cm long x 2.3 cm o.d., with 30 inserted pieces of polypropylene tubing (Jacob Bros. Sepulveda, Calif.) 47 cm long with 3.2 mm o.d. and 1.6 mm i.d., which were cut obliquely at the ends. Before insertion of the nitrogen lines into the tubes containing the samples, the lines were vigorously shaken, 641

0003-2697/78/0872-O&1$02.00/0 Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

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and passage of nitrogen was initiated at a rate sufficient to be detected by moistened lips. After placing the nitrogen lines in all sample tubes, the extra lines are submerged to a water head of several inches. The above-described procedure will cause 24 tubes to bubble simultaneously. After use, the nitrogen lines were not allowed to dry but were continuously soaked in water. The nitrogen lines were cleaned by changing the water and by wiping with a Kimwipe several times at several-hour intervals. The test tubes (16 x 9%mm polypropylene tubes) obtained from Van Waters and Rogers (Los Angeles, Calif.) were marked at a volume corresponding to 2.4 ml. The Ellman’s reagent [5,5’dithiobis(2nitrobenzoic acid)] was from Pierce Chem Co. (Rockford, Ill.), and the urea, guanidine HCl (grade l), and disodium EDTA were from Sigma Chemical Co. (St. Louis, MO.). Higher cysteine values were achieved when the urea solution was prepared daily; this may be due to the presence of ammonium cyanate, an interconvertible isomer of urea (8) which would undergo rapid nucleophilic addition with thiols. The sodium borohydri$e was from Metal Hydrides, Beverly, Mass. PROCEDURES

Between 4 and 5 mg of seed meal weighed with a Cahn electrobalance was placed in 16 x 9%mm polypropylene tubes premarked at 2.4 ml. Approximately 5 ~1 of octanol was added to control foaming. Several hours before use, a quantity of 3 mM EDTA-8 M urea (35 mg disodium EDTA and 16.8 g of urea/35 ml) sufficient for all samples was prepared. Immediately before use, sodium borohydride was dissolved in EDTAurea solution (280 mg/35 ml) to give an 8 mgiml solution. The resultant 8 mg/ml NaBH,-8 M urea-3 mM EDTA solution was pipetted (1.2 ml) with a j-ml pipette into the polypropylene centrifuge tubes. The tubes were vortexed so that the solution wetted any seed powder near the top of the tubes and were placed in a 38°C shaking water bath for 1 hr. At approximately 20-min intervals the tubes were again vortexed and gently rotated to cause the larger particles to settle to the bottom of the tube. After the incubation period, destruction of the sodium borohydride was begun by addition of 0.2 ml of 1 M KH,PO, in 0.2 N HCl, followed by vigorous vortex mixing. Five minutes after addition of the acidic phosphate solution, the tubes were again vortexed. Acetone was added (0.8 ml) with a repipette, and, after a piece of Parafilm was placed over the tops, the tubes were vigorously inverted. Nitrogen bubbling with the previously described device was carried out for 5 min, after which 0.2 ml of a 4 mgml acetone solution of Ellman’s reagent was added directly to the bubbling tubes. Four minutes after the Ellman’s reagent was added to all tubes, the bubbling line was removed from the tube which first received Ellman’s reagent and was

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643

submerged several inches in a water reservoir so as not to “short circuit” the nitrogen supply. Glass-distilled water was added to adjust the volume to the premarked, 2.4-ml level, and the procedure was continued for the remaining tubes. The tubes were again vortexed and then centrifuged at 10,OOOg in a 24-hole rotor (Sorvall-SM24) in a Sorval RC2-B centrifuge for 10 min. After centrifugation, approximately 1 ml of the supernatant fluid was carefully removed, with care being taken to avoid the extremely flocculant pellet which can cause turbidity problems. The absorbances were determined in a Coleman 111 spectrophotometer at 411 nm using a l-ml quartz curvette. Probably because of the unusual viscosity of the 8 M urea-acetone solution, reproducible absorbances were difficult to obtain with a Gilford 240 autosampler. Two kinds of blanks were initially prepared. One blank contained Ellman’s reagent but no seed meal, and the other blank contained seed meal but no Ellman’s reagent. The former blank corrected for residual NaBH,, which would reduce the Ellman’s reagent to give the anionic chromophore, and typically had a value of 0.17 absorbance unit. The latter blank corrected for sample turbidity and original seed color. Sodium borohydride bleaches seed meal color (probably by reducing aldehydes and ketones on seed meal chromophores to alcohols); and, since turbidity was fairly constant, this blank was omitted and replaced with an empirically derived value of 0.008 absorbance unit/mg of seed meal. The cysteine concentration was then calculated using the molar absorptivity of 12,000, as previously reported (6), after subtracting the value of the blank containing Ellman’s reagent but no seed meal and subtracting the value of 0.008 absorbance unit/mg of seed meal to correct for sample turbidity and original color. RESULTS

AND DISCUSSION

The correspondence between cystine values determined colorimetritally and by performic acid oxidation is rather good (Fig. 1). Not only are the calorimetric and cysteic acid-determined values proportional, but in many cases the values lie along the theoretical line representing a I:1 correspondence of absolute values. As can be seen from the regression equation (y = mx + b) coefficients listed in Table 1, the correspondence is especially good for peas and lentils, and peas plus lentils plus beans, but not for fava beans. In Table 2 are listed the actual values determined in this study and in the previous study (7). The lower than expected values for beans and fava beans have been repeated approximately six times. We have examined the time course for the sodium borohydride reduction and for color formation to elucidate the cause of the low calorimetrically derived cystine values.

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A

0 Beans Lentils A Fobo beans 0 Peas l

ioy,,,,, 0.010

CYSTINE:

0.015

BY PERFORMIC

0.020

ACID

0.025

/

0.030

OXIDATION-ION (mmoles/g meal)

0.040

EXCHANGE

CHROMATOGRAPHY

FIG. 1. Comparison of cystine values derived by NaBH, reduction followed by thiol estimation with Ellman’s reagent and by performic acid oxidation/ion-exchange chromatography. The values were taken in duplicate. The line represents what would be an ideal 1: 1 correspondence between the two methods.

For the fava bean Diana we have found that 20 and 35 min of sodium borohydride reduction yields 84 and 95%, respectively, of the cystine values obtained at 1 hr of reduction. A time course for the color development step showed a 17% increase (0.027 absorbance) in the blank (minus meal plus Ellman’s) 72 min after centrifugation but less than a 1% change in the color of the legume seed meal after correction for increase in background color development. TABLE

1

COEFFICIENTS FOR REGRESSION EQUAT~ONY DETERMINED COLORIMETRICALLY(~)

Beans Fava beans Peas Lentils Peas + lentils Peas + lentils + beans Peas + lentils + beans + fava beans

= mx + b RELATING CYSTINE AND BY CYSTEIC ACID(X)

Slope

Intercept

m

b

.64 .59 .92 .88 .94 .99 .70

.OOl .004 .002 ,001 ,001 .OOl ,005

Correlation coefficient .99 .87 .92 .96 .91 .89 .75

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OF CYSTINE

VALUES

DETERMINED

2 COLORIMETRICALLY

AND

AS CYSTEIC

ACID

Cystine (mmolig of meal) Speciesicultivar Beans Bean Pinto Beans Fava beans Tarvin Tick Primeperle Diana Klein Thuringer Herz Freyz Erfordia Peas Line No. 1

?

i 5 6 7 9 75 76 78 84 87 89 90 91 93 95 98 90 Lentils Tekoa Morden Amasaya Eskiseher Pioner Red Slovens Kamadra (’ The value given in original publication

Cysteic acid (7) determination

Calorimetric determination

,017 ,021

.012 .013

.030 ,032 .041 .026 ,025 .023

.025 ,022 ,022 .023 .023 .022

.024 ,031 ,028 .028” .030 ,030 ,029 .025 .024 .022 ,024 ,023 ,022 ,024 .031 .022 .024 ,020 .023

,024 ,031 .029 .030 ,030 ,031 .028 ,023 .024 ,020 ,028 ,022 .023 .024 .022 .024 ,022 ,023 ,023

,020 .018 ,021 ,021 ,023 .019

,019 ,017 .020 ,019 .017 .020

(7) was in error.

It is to be noted that in the original paper describing use of sodium borohydride in 8 M urea, theoretical values were obtained for five out of six proteins, with chymotrypsinogen yielding 80% of the theoretical value (6). Later work on the comparative ease of disulfide reduction in chymotryp-

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sinogen showed that one of the five disulfide bonds was more refractory to disulfide reduction than the others (9), which may explain why Cavallini et al. (6) effectively achieved disulfide reduction of only four of the five disulfide bonds. It is to be noted that reduction of all five disulfide bonds of chymotrypsinogen with the sodium borohydride has been reported in pH 9.6, buffered 8 M urea (10). Since disulfide bonds in a few proteins are resistant to reduction in 8 M urea but easily reduced in 6 M guanidine hydrochloride (1 1), the Diana seed meal was subjected to NaBH, reduction in 6 M guanidine HCI, and a value of 0.027 mmol/g of meal was found. This value compares to 0.022 mmoYg of meal in the 8 M urea but is still considerably lower than the 0.041 value previously reported (7). The ability of 6 M guanidine HCl to yield cysteine values closer to those previously reported (7) appears to be lot-specific and is the subject of a present study on a large sample ofPhaseolus vulgaris. An unexpected advantage of the 6 M guanidine HCl is that the pellet is much less flocculant than in the urea, so that sample turbidity is markedly reduced. Leach and Fish (12) found that 6 M guanidine. HCI took 3 weeks to completely denature soybean trypsin inhibitor at 25°C but only 15 min at 70°C. Accordingly, the fava meal was subjected to 6 M guanidine*HCl 8 mg/ml NaBH, for 1 hr at 60°C. The unexpected result was that the 60°C treatment gave a lower cysteine value than the 38°C I-hr treatment. It is possible that the low cysteine values obtained at 60°C are the result of rapid cysteine reoxidation and NaBH, heat instability. Experiments in which the EDTA concentration was varied from 0 to 50 mM and in which the time between the acid-NaBH, quench to acetone addition was varied from 5 to 20 min showed that the conditions used here were optimal. Should legume seed meals be shown to be easily reducible, or to be highly refractory, it would seem necessary to determine the nutritional availability of both classes of disulfide bonds. In conclusion, we feel that the described calorimetric assay is certainly capable of being used as a cystine screening assay in peas and lentils, and perhaps in beans and fava beans. Experiments that will study a larger sample of beans are now in progress. ACKNOWLEDGMENTS We thank Joyce McLean, manuscript preparation.

Susanna Kearns-Sharp.

and Jan Moore for assistance in

REFERENCES 1. 2. 3. 4. 5.

Autret, M. (1972) FAO Nutr. Stud. 24, 213-229. MacKenzie, S. L. (1977) J. Chromutogr. 130, 399-402. Moore, S. (1963) J. Biol. Chrm. 238, 235-237. Goa. J. (1961) Acra Chem. Scund. 15, 853-855. Zahler, W. L.. and Cleland, W. W. (1968)J. Biol. Chem.

243, 716-719.

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6. Cavallini, D., Graziani, M. T., and Dupre, S. (1966). Nature (London) 212, 294-295. 7. Bhatty, R. S., Finlayson, A. J., and MacKenzie. S. L. (1977) Canad. J. PIunr Sci. 57, 177-183. 8. Friedman, M. (1973) The Chemistry and Biochemistry ofthe Sulfhydryl Group in Amino Acids, Peptides, and Proteins. p. 106, Pergamon Press, New York. 9. Sondack, D. L.. and Light, A. C. (1971) J. Biol. Chem. 246, 1630- 1637. 10. Light, A.. and Sinha, N. K. (1967) J. Biol. Chem. 242, 1358-1359. 11. Shapira, E., and Arnon, R. (1969) J. Biol. Chem. 244, 1026-1032. 12. Leach. B. S.. and Fish, W. W. (1977) 3. Biol. Chem. 252, 5239-5243. PETER FELKER GILES WAINES Department University Riverside, Received

of Plant Sciences of California Californiu 92521 August 29, 1977; accepted

Februury

23. 1978