An isotopic method for determining chemically reactive lysine based on succinylation

J . Sci. Food Agric. 1984,35,472-480

An Isotopic Method for Determining Chemically Reactive Lysine Based on Succinylation

Trevor R. Anderson' and George V. Quickeb

Department of Biochemistry, University of Zululand, PIBagX1001, Kwa- Dlangezwa, 3886 South Africa and "Department of Biochemistry, University of Natal, PO Box 375, Pietermaritzburg, 3200 South Africa

(Manrrscriptreceived 19 May 1983)

A method is described for the routine succinylation of proteins using [i4C]succinic anhydride as a means of measuring free .+amino groups. Maximal succinylation was achieved using 6~ guanidine hydrochloride as protein solvent and an 80-fold molar excess of succinic anhydride relative to total lysine residues. Treatment with hydroxylamine (pH 13, 25"C, 5 min) removed unwanted 0-succinyl esters. Succiny- lated protein was precipitated with tnchloroacetic acid, residual label washed out with ethanol and the extent of labelling measured in a scintillation counter. The method gave close to theoretical values (n.s., P>0.05) for lysyl residues in egg white lysozyme, bovine haemoglobin, ovalbumin and bovine serum albumin but gave a low value with /3-lactoglobulin and an overestimate with insulin attributable to the relatively high contribution of a- relative to &-amino groups in this low molecular weight protein. The method gave good results for 12 soya protein samples and was shown to be very sensitive to 'isopeptide-type' heat damage in these samples. The results correlated well ( r = 0.91) with those obtained by the well established dye-bound lysine difference procedure whereas, as could be expected, both these methods correlated poorly with total lysine determinations ( r = 0.69 for succinic anhydride and r = 0.77 for the dye-binding method). The proposed method shows promise as a rapid procedure for the estimation of available lysine, but further studies are necessary to test its ability to measure nutritionally available lysine in all categories of heat damage.

1. Introduction

Lysine, the most limiting essential amino acid in many foods of plant and animal origin, because of its highly reactive &-amino group is particularly susceptible to a variety of side reactions which result from excessive heat treatments in cooking and industrial processing. Heat treatment may be responsible for the formation of poorly digestible lysine isopeptides as well as unavailable lysine derivatives of carbohydrates, lipids, nucleic acids, polyphenols and vitamins, one or more of which occur prominently in most protein foods. Furthermore, commonly used food preservatives and additives such as sodium bisulphite, formaldehyde and alkali cause substantial reductions in nutritionally available lysine.'.' Many of the end-products have also been reported to be t o x i ~ . ~ - ~

A number of procedures have been developed in attempts to assess accurately the nutritionally available lysine. The tedious in-vitro methods are now becoming less popular and being replaced by simpler, more rapid chemical assays mainly utilising E-amino-reactive reagent^.^ Although no single method has proved completely acceptable the so-called dye bound lysine (DBL) difference procedure has been semi-automated.*

Succinic anhydride, a non-volatile, reasonably stable solid, has been widely used for the chemical modification of purified proteins for the purposes of studying a variety of properties and structural aspects.'It is known to preferentially react with a-amino and &-amino groups" (Figure l(a)) but has not been used for specific amino group modification because it also simultaneously forms 0-succinyl esters of serine, threonine and tyrosine" (Figure l(b-d)), S-succinyl esters of cysteine'* (Figure

Present address: Department of Biochemistry, University of Natal, PO Box 375, Pieterrnaritzburg, 3200 South Africa.

472

Determination of chemically reactive lysine 473

(cH,) ,-%H~

CH2-OH

pH > 7

- C H 2 0 - H

.CH2-SH

F H 3 CI - C-0-C(CH, ),-COO- H

NH,OH

pH 13, 25OC __c__)

0

. C H 1 Ph-O-k!-(CH,),--COO-

- NO reaction

H O \ 11

-CH2-OH + y-C-(CH2) ;-COO-

Ho Succinohydroxamate

y3 "\ B . C-OH + N-c-(cH,),-coo- H /

HO

H o >N-!! -(CH,)+Od

HO

+ -CH,Ph-OH

H 8 \N-c-(cH,),- COO- /

HO - CH ,-SH +

H R \N-c-(cH,),- coo- HO'

6OC-(CH,) ,-COO-

Figure 1. Succinylation followed by reaction with alkaline hydroxylamine of protein-bound lysine (a), serine (b), threonine (c). tyrosine (d), cysteine (e), and histidine (f). 0-succinyl tyrosine. S-succinyl cysteine and possibly succinylimidazole derivatives also desuccinylate spontaneously within 3-4 h to give free succinic acid residues.".'2 Ph= phenyl.

474 T. R. Anderson and G. V. Quicke

l(e)) and succinylimidazole derivatives of histidine13 (Figure l(f)). The fact that the tyrosyl and cysteinyl esters desuccinylate spontaneously within 3 -4 h,", l2 and that alkaline hydroxylamine deacylates all the above esters (Figure 1) whereas under these conditions the N-succinyl groups are stable," prompted the idea of using [14C]-succinylation followed by treatment with alkaline hydroxylamine, precipitation with trichloroacetic acid and liquid scintillation counting to estimate reactive lysine residues in protein material.

This report describes the development of the assay procedure, its application to six model proteins and its comparison with the more established DBL difference method' using 12 differentially heat damaged soya protein samples.

2. Materials and methods

2.1. Model protein solutions Crystalline bovine pancreatic insulin (2.41% H20), egg white lysozyme grade I (1.09% H20) , bovine haemoglobin Type I (2.88% HZO), milk /3-lactoglobulin thrice crystallised (1.51% H,O), ovalbumin grade V (2.36% H20) and bovine serum albumin (0.15% H20) were obtained from Sigma Chemical Company, Saint Louis, U.S.A. Test solutions (1 to 2 m ~ in the case of insulin and lysozyme or 0.2 to 0 . 5 m ~ for the other proteins) were prepared by dissolving suitable quantities of protein in 6~ guanidine hydrochloride.

2.2. Soya protein samples Isolated soya protein (Brand Purina Assay Protein RP100) was employed in these studies and found to contain on a dry matter basis 96% 'crude' protein, 1.4% ash, 0.3% fat, 0.2% 'crude' fibre and 2% N-free extracts. This material was used to prepare 12 samples in which 80 parts of soya protein and 20 parts distilled water were heated in airtight metal containers at different temperatures (90, 110, 130°C) for different periods of time (0.5, 1, 2, 4 h).

2.3. Moisture, 'crude' protein and total lysine (TL) Moisture was determined as the loss in mass after heating in a hot air oven at 130°C for 3 h. Total nitrogen was determined by macro-Kjeldahl (potassium sulphate-mercuric oxide catalyst), and protein estimated as N X 6.25. Total lysine values were obtained from conventional amino acid analyses following digestion in V ~ C U O with 6~ HC1 (llo"C, 24 h) using a Beckman 119C or a Biotronik BC 2000 Amino Acid Analyser.

2.4. ['4C]-succinic anhydride Initially [2,3-'4C]-succinic anhydride was prepared from [2,3-'4C]-succinic acid and acetic anhydride according to a published p r ~ c e d u r e . ' ~ The purity of the product was confirmed by melting point (119.7- 120°C) and 'mixed' melting point determinations (Buchi SMP-20 apparatus) and the i.r. spectrum in pure solid potassium bromide (1 : 50 wiw), which gave characteristic bands at 1840 and 1784 cm-' (C=O stretch), 1320-1290 cm-' and 1140 cm-' (C-0-C stretch) and 1000 cm-' (C-0-C stretch in 5-membered ring anhydrides). Two batches of material (specific activity 3.76 x pCi pmol-') were used in the experiments.

[1,4-'4C1-succinic anhydride (specific activity 105 pCi pmol-' ; radiochemical purity 97.5%) was obtained from the Radiochemical Centre, Amersham, England. This material (250 pCi) was diluted with unlabelled succinic anhydride (Merck, GR grade) to give a specific activity of 2.60 X FCi pmol-', dissolved in a minimum volume of hot redistilled acetic anhydride (b.p. 139- 140°C), crystallised by cooling in ice, filtered, washed with sodium-dried ether and rapidly dried in a vacuum desiccator. The presence of pure succinic anhydride was confirmed as described above.

and 6.80 x

2.5. Succinic anhydride (SA) reactive lysine (method A) Succinylation of the model proteins was carried out according to the procedure of Gounaris and Perlmann" with certain modifications. Three 20 ml aliquots of each protein solution were


succinylated at pH 8.0 and 18°C with an 80-fold molar excess of [2,3-'4C]-succinic anhydride (specific activity 3.76 X pCi pmol-') over estimated moles of lysine, employing 4~ NaOH to maintain constant pH (Metrohm-Herisau Combi 5 pH-stat system). The anhydride was added as a series of small portions; each portion was added as base uptake slowed down. Three 1 ml aliquots of each succinylated protein solution were then treated in a 15-ml centrifuge tube with 2 ml of alkaline hydroxylamine reagent (pH 13.0) at 25°C for 5 min, the protein precipitated at 5% trichloroacetic acid and centrifuged for 5 min in a bench-top centrifuge. The supernatant was removed by decantation (taking care not to lose any unsedimented material) and the pellet washed four times with 2 ml aliquots of ethanol (reagent grade). The final precipitate was dissolved in 1 ml of 0 . 2 ~ NaOH, mixed with 10 ml of Insta-gel scintillator (Packard Instrument (Pty) Ltd), poured into a 20-ml low-potassium glass vial and counted for 1 min (or a minimum of 10 000 counts in channel 1) in a Packard Tri-Carb Model 3003 Liquid Scintillation Spectrometer. The counting efficiency for each sample was read from a quench correction curve for the Channels Ratio Method. In the case of low-counting efficiencies which were due to colour quenching (e.g. for haemoglobin), the procedure of Neame" was employed. This yielded a mean counting efficiency for haemoglobin of approximately 65% which compared well with efficiencies obtained with the other protein samples (70-75%) and for unquenched [methyl-'4C]-toluene standard (80.5%).

2.6. Succinic anhydride (SA) reactive lysine (Method B) A simplified procedure was developed in order to overcome the problem involved in taking representative aliquots of insoluble suspensions, to minimise the transfer of reacting materials and to avoid the need for using a pH-stat. Duplicate 12-15 mg portions of each sample were accurately weighed into 20-ml glass scintillation vials, a 10 mm magnetic stirring bar added, followed by 3 ml of 6~ guanidine hydrochloride, the mixture being stirred at 50°C for 30 min. After cooling to room temperature (ca 20"C), 0.1 ml of 5 M NaOH was added and solid [1,4-14C]-succinic anhydride (specific activity 2.60 x pCi pmol-') was added in 7-mg portions over a period of 30-45 min to give an 80-fold molar excess over total lysine content. Vigorous stirring was maintained throughout the process and a further two aliquots (0.1 and 0.05 ml) of 5M NaOH were added after ca 15 and 30 min respectively. The temperature was raised to 25-30°C 10 min after the last portion of succinic anhydride had dissolved, and 5 ml of alkaline hydroxylamine reagent (pH 13) was added. The mixture was stirred for exactly 5 min, the stirring bar removed and the protein precipitated at 5% trichloroacetic acid. The vials were capped, lowered into Schott-Mainz 50-ml centrifuge tubes, suitably cushioned with plastic polytops and centrifuged for 5 min, the supernatant carefully decanted and the precipitate washed twice with 10 ml aliquots of absolute ethanol. The washed precipitate was then treated with 1 ml of 0.2M NaOH, employing heat (if necessary) to dissolve the protein or, in the case of the soya samples to achieve a fine homogenous suspension of the insoluble material. Insta-Gel(l0 ml) was then added to each vial, stirred vigorously and counted as described in Method A above. It was not necessary to use a solubiliser with the soya protein samples tested. Coloured solutions were treated according to Neame's procedure (see section 2.5).

2.7. Effectiveness of hydroxylamine reagent for the removal of 0-succinyl groups [2,3-'4C]-succinyl-1ysozyme was prepared from 2 m ~ lysozyme and [2,3-'4C]-succinic anhydride (specific activity 3.76 x pCi pmol-') using Method A and then left standing for 4 h to allow for the spontaneous decomposition of 0-succinyl tyrosine, S-succinyl cysteine and the succinylimidazole derivative of histidine. A series of hydroxylamine solutions of pH values 7,9,10,11,12,13 and 13.68 were prepared by titrating 20 ml of 2M hydroxylamine hydrochloride (Sigma Chemical Company (Pty) Ltd) to the desired pH with 3 . 5 ~ NaOH. Aliquots (1 ml) of succinyl-lysozyme were mixed in separate 15-ml glass centrifuge tubes with 2ml of each hydroxylamine solution and incubated at 25°C for 5 min, using distilled water instead of hydroxylamine reagent in the control treatment. After incubation the protein was precipitated at 5% trichloroacetic acid and treated further as outlined for Method A.


100

80

- I z 5 60 .- .- - =- .-

I 40

n ?

2 0

0

2.8. Ability of ethanol to remove residual label from trichloroacetic acid precipitates [2,3-14C]-Succinyl-insulin was prepared from 2 m ~ insulin and [2,3-'4C]-succinic anhydride (specific activity 6.8 x'10T4 pCi pmol-') according to Method A. A 1 ml aliquot of succinyl-insulin was then treated with 2 ml of alkaline hydroxylamine reagent (pH 13.0) and the protein.precipitated with 4.0 ml of 10% trichloroacetic acid. After centrifuging and before decantation two 1 ml aliquots of the supernatant were removed and each mixed with 10 ml of Insta-Gel and the radioactivity measured. The protein precipitate was then washed with four successive 2 ml aliquots of ethanol followed by one 2 ml aliquot of diethyl ether (reagent grade) to remove any residual quenching agents; 1 ml aliquots of the supernatant were removed for counting after each washing.

2.9. DBL difference procedure The soya protein samples were analysed according to the manual method of Hurrell et aL8

- x/x-x

Figure 2. Effect of pH of hydroxylamine reagent (25"C, 5 min) on the removal of 0-succinyl groups from the serine and threonine residues in succinylated egg white lysozyme. Values, expressed as a percentage of desuccinylation at pH 13, are the means of duplicate

~ ~ 1 1 , 1 ~ 1 , , determinations.

X/X x-/ 7 8 9 10 I I 12 13 14

3. Results and discussion

3.1. Conditions for maximal &-N-succinylation In order to denature the proteins, 6~ guanidine hydrochloride was used as protein solvent for the succinylation process. Although this increased the extent of E-N-succinylation, relatively high quantities of anhydride were required in order to achieve maximal succinylation. Preliminary work showed that whereas bovine haemoglobin required only a 53-fold molar excess for apparent maximum N-succinylation, ovalbumin needed a 67-fold molar excess, and lysozyme, P-lactoglobulin and bovine serum albumin an 80-fold molar excess of anhydride over estimated lysine content. On these grounds it was decided to use the 80-fold level for all subsequent analyses.

3.2. Effectiveness of hydroxylamine reagent for removal of aliphatic 0-succinyl groups Gounaris and Perlmann, who reported 100% N-succinylation of active pepsinogen but only 15-20% 0-succinylation of its serine and threonine residues, found that neutral hydroxylamine speeded up the decomposition of 0-succinyl-tyrosine residues but had no effect on the 0-succinyl aliphatic esters. It was therefore important to establish optimal conditions for effective de-0-succinylation.

pH of hydroxylamine reagent


Egg white lysozyme with a relatively high ratio of aliphatic hydroxy aminoacyl residues relative to lysine residues (10 Ser, 7 Thr, 6 Lys) was chosen as a suitable model.

Probably because of the large excess of anhydride employed in the present study, as much as 72% of the total aliphatic hydroxy amino acid residues were succinylated, which further emphasised the importance of maximal de-0-succinylation. Figure 2 shows that for reagent pHvalues of 7- 11, there was a small amount of de-0-succinylation of succinyl-lysozyme during the 5 min incubation period (10.2% at pH 11). The extent of desuccinylation rose sharply to 97.1% at pH 12reaching a maximum at pH 13. These results imply that the rate, if not the extent, of de-0-succinylation of aliphatic esters with hydroxylamine is strongly pH-dependent. Similar results have been recorded for the reactivity of hydroxylamine with carbonyl-containing haems, to form oximes. l6

It should be noted that a 2~ hydroxylamine reagent (pH9) has been reported to cleave asparaginyl-glycine bonds and, to a lesser extent, asparaginyl-leucine bonds in reduced carboxymethylated bovine pancreatic ribonucleases owing to cyclisation of asparaginyl side chains leading to the formation of intrachain cyclic imides which are susceptible to nucleophilic attack by hydro~y1amine.l~ These findings however, would only affect the present studies if cleavage of these bonds yielded peptides that were not precipitated by trichloroacetic acid. Such losses would probably be minimal since this cleavage is highly specific. Furthermore. Bornstein" found that cleavage was retarded at pH values greater than 10.5 owing to competitive nucleophilic attack by hydroxyl ions, leading to saponification of the cyclic imides. This constitutes a further advantage in using a hydroxylamine reagent of pH 13.

Table 1. Ability of ethanol to remove residual label ([2,3-'"C)- succinic acid and [2,3-14C]-succinohydroxamate) from trichloro-

acetic acid precipitates of succinyl-insulin

Label removed Source of l4c Counts

counted min-'" Protein bound label

Decanted with TCA 434770 101.09 1st ethanol wash 6518 1.52 2nd ethanol wash 394 0.09 3rd ethanol wash 60 0.014 4th ethanol wash 86 0.02 Ether wash 12 0.003 Succinyl-insulin 4301 -

a Values are means of duplicate determinations.

3.3. Ability of ethanol to remove residual label from trichloroacetic acid precipitates Although trichloroacetic acid proved to be an effective precipitant for the succinylated proteins studied here (Tables 2 and 3) all residual label, comprising ['4C]-succinic acid and ["CC]- succinohydroxamate residues (Figure l), could not be removed by washing with trichloroacetic acid. It was therefore necessary to find a suitable solvent for these two compounds which would not dissolve any protein precipitate. Ethanol and acetone are solvents for succinic acid'' but in the absence of information about the solubility of the succinohydroxamate in these solvents, their suitability as washing solvents had to be tested experimentally. As seen in Table 1 the bulk of the residual label is removed when the trichloroacetic acid supernatant is decanted, and ethanol efficiently removes most of the remaining label within the first two washings. The third and fourth ethanol washes respectively removed only 1.4 and 2.0% of labelled material relative to lysine-bound label. Omission of these washes would therefore result in minimal overestimation of SA-reactive lysine. The final wash with ether was introduced as a means of removing any quenching agents that might still be present. In order to cover situations in which the washing procedure might be less efficient, it is suggested that four ethanol washings would be suitable. An overall recovery of 99.4% of the label used for the succinylation reaction substantiated the validity of the procedure used.

478 T. R. Anderson and G . V. Quicke

3.4. Succinylation of model proteins Initially the six proteins were analysed by Method A. After Method B was developed for use with feed samples, the model proteins were reanalysed using this procedure. The results presented in Table 2 clearly show that there was no significant difference ( P > 0.05) between the values obtained with the simpler Method B and the more cumbersome Method A. Both methods measured lysine in each protein similarly and the results were highly reproducible. These results validated the use of Method B in the subsequent soya protein analyses.

Table 2. Succinic anhydride @A)-reactive lysine and total lysine content of six model proteins.'

SA-reactive lysine

Protein

Method A Method B Total lysine

Mean (n = 3) Mean (n=2) Mean (n = 2)

Bovine insulin 4.36 4.26 2.50

Bovine haemoglobin 10.2Ob 10.16' 10.71 P-Lactoglobulin 8.06 8.22 10.35 Ovalbumin 6.33 6.32 6.11 Bovine serum albumin 11.01 10.80 11.24

s . e . (mean) 0.05 0.08 0.24

Egg white lysozyme 5.91 5.73 5.51

Results are exprcssed in g lysine per 100 g air dry sample. ' Haemoglobin values were corrected for a 5% loss of radioactive label during the

bleaching process as recommended by Neame .I5

When, however, the values obtained for Method B were compared with the corresponding total lysine values (Table 2), analysis of variance showed the interaction term (methods X proteins) to be significant ( P < O . O O l ) indicating that the two methods did not give similar values for every protein. Indeed, Method B gave a good, reproducible estimate (Table 2) of the lysine residues present in four of the six proteins tested. The high degree of succinylation of &-amino groups achieved is best demonstrated with ovalbumin which has no a-amino groups since the N-terminal glycines are acetylated. By contrast, the values obtained for insulin and P-lactoglobulin were not satisfactory as they were, respectively, over- and underestimated. Insulin may represent an extreme case as it contains a high proportion of a-amino groups relative to its molecular mass and to &-amino groups ( 2 : 1) and it was found (Anderson, T. R., unpublished work) that the a-amino groups of free alanine and of glycyl-lysine were 95% succinylated despite efforts to minimise a-amino succinylation by varying the pH and temperature of the reaction medium. Inefficient removal of 0-succinyl groups from the insulin molecule or contamination of the precipitated protein with residual label are discounted in view of the demonstrated effectiveness of the hydroxylamine reagent (pH 13) as de-0-succinylation agent and of ethanol as washing solvent as well as the satisfactory values obtained with four of the model proteins and the soya protein meals. As the contribution of a-amino groups is likely to be small in the case of complex food proteins, there appears to be no need to correct for this. However, for products known to contain a large proportion of a-amino groups, some correction may be necessary.

Under-estimation of the lysyl residues in P-lactoglobulin is more difficult to explain. It seems unlikely that the crystalline protein contains 20% unreactive lysyl groups, nor does itseem likely that succinylation was only 80% effective as an 80-fold molar excess of succinic anhydride relative to total lysyl residues gave close to 100% E-succinylation for the other model proteins. However, since succinylation is reported to render proteins more soluble in polar solvents2' it is possible that some of the precipitated S-b-lactoglobulin may have been lost in the ethanol washes. A less polar solvent, acetone, was accordingly used to wash the trichloroacetic acid precipitates, but use of this solvent


gave an overestimation of lysyl residues by ca 15%. This may have been because of poor solubility of succinohydroxamate in this solvent and the use of other non-polar solvents warrants investigation. The possible use of millipore filters which would retain all soluble labelled protein and thus eliminate both the need for trichloroacetic acid precipitation and specialised washing solvents is receiving attention.

3.5. Analysis of soya protein samples Table 3 shows that all three methods give at least some measure of sensitivity to lysine damage and that, generally, increased severity of heat treatment resulted in a corresponding decrease in reactive lysine. The D B L and S A methods however were considerably more sensitive than the T L method, particularly for the most severely damaged samples. This is reflected in the relatively low correlation between TL values and both D B L values ( I = 0.77) and SA values ( I = 0.69). Since the predominant lysine damage in these soya samples is probably isopeptide crosslinks with glutamate and aspartate residues,' overestimation of available lysine by the T L method can be expected as isopeptides are acid-labile and hence all lysine in this form is measured by total amino acid analysk2' By contrast the SA method correlated very well with the well established DBL procedure ( r = 0.91) and gave very similar absolute values.

Table 3. Succinic anhydride reactive lysine (SA) values for differentially heat damaged soya protein samples compared and correlated with those obtained by the Total Lysine (TL) and Dye

Bound Lysine Difference (DBL) Procedures."

Sample TL DBL SA

Soya 90°C 0.5 h 1.0h 2.0 h 4.0h

110°C 0.5h 1.0h 2.0h 4.0h

130°C 0.5h 1.0h 2.0h 4.0 h

6.5 5.9 5.6 5.3 6.2 5.8 5.0 5.1 6.0 5.8 4.6 4.8

5.9 5.1 5.7 5.8 5 . 2 5.7 4.9 5.4 5.4 5.4 5.1 5.4 5.2 5 .2 4.5 4.9 5.3 4.6 4.2 4.8 3.6 3.1 3.0 2.6

s.e. (mean) 0.14 0.05 0.11

Results are expressed in g lysine per 16 g N and are means of duplicate determinations.

4. Conclusions

The proposed isotopic procedure (Method B) for evaluating chemically-reactive lysine residues in food protein offers a new, relatively rapid (3 h) procedure which can readily be applied for routine analysis of the 'available' lysine content of food samples. Application of the modified procedure (Method B) to crystalline proteins gave values in very close agreement with those obtained by the procedure which provides for more rigorous control of p H in a pH-stat (Method A). The values obtained for four of the six model proteins compared satisfactorily with total lysine values as determined by amino acid analysis, but low values for P-lactoglobulin could not be satisfactorily explained. The method gave good results for soya protein samples and was shown to be very sensitive to the effect of heat damage in these samples. The results compared favourably with those obtained by the well established DBL difference method.

It should however be stressed that Hurrell and Carpenter" found that both bound and FDNB-reactive lysine in severely heated proteins were digested and absorbed to the same extent.


They therefore warned against assuming that reactive lysine will always be equivalent to nutritionally available lysine. To establish the full validity of the proposed procedure, further studies are in progress to test its ability to measure nutritionally available lysine in other categories of heat damage occurring in both plant and animal materials (Erbersdobler, H.; Anderson, T. R.; unpublished).

Acknowledgements The authors are indebted to Mr W. Nel (Department of Biochemistry, University of Zululand, Republic of South Africa) and Dr J. Mathers (Department of Applied Biology, University of Cambridge) for helpful discussions; the Council for Scientific and Industrial Research for financial support; Prof Dr H. Erbersdobler (Institute for Human Nutrition and Food Science, University of Kiel, German Federal Republic) for providing’the soya protein material.

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3.

4.

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An isotopic method for determining chemically reactive lysine based on succinylation