mechanisms of heat damage in proteins

8/6/2019 Mechanisms of Heat Damage in Proteins

1/17

B r . J . Nutr. (1970), 24, 3 1 3 3 = 3

Mechanisms of heat damage in proteins2." Chemical changes in pure proteins

BYJ. BJARNASON? AND K. J. CARPENTERDepartment of Agricultural Science and Applied Biology,

University of Cambridge(Received 31 July 1969-Accepted 25 September 1966)

I . Bovine plasma albumin (BPA) containing approximately 14% moisture, when heated for27 h at I I ~ O uffered an appreciable loss of cystine and a small loss of lysine; at 1 4 5 ~ll theamino acids except glutamic acid and those with paraffin side-chains, showed considerablelosses. Isoleucine also showed some loss through racemization to alloisoleucine.

2. BPA heated at I 1 5 ~volved H2S;at 145' other sulphur compounds were released as well,all coming from the breakdown of cystine. Possible mechanisms for this are discussed.3. Ammonia was also liberated from BPA heated a t I 5'. The degree of correlation of lysinebinding in different proteins with ammonia liberation and amide changes has led us to suggestthat the main reaction of -amino lysine groups is with amide groups of asparagine and gluta-mine. Reaction of e-amino groups with carboxylic groups is thought to be less important.4.Model experiments have shown that a reaction between amide groups and the e-aminogroup of lysine in proteins can occur at practical drying temperatures.5 . Reactions of the e-amino group of lysine with destruction products of cystine is also

considered to be partially responsible for the lysine binding in heated proteins.

It has long been known that the Maillard reaction between sugar aldehyde groupsand the free amino acid groups of proteins can explain much of the damaging effectof heat on the protein quality of dried milk. However, heat damage can also occurif meat or fish products are heated excessively and these are essentially carbohydrate-free. This too is accompanied by a loss of reactivity of the s-amino groups of lysine.Any oxidizing fat present may contribute carbonyl groups for a Maillard-type reactionbut high-temperature heat damage cannot be considered as being just due to reactionwith other non-protein compounds present, since it will occur to the same extent withpure protein preparations (Carpenter, Morgan, Lea & Parr, 1962). The changes alsooccur in an atmosphere of nitrogen as well as of oxygen (Carpenter, Ellinger, Munro& Rolfe, 1957).This study was designed to investigate the chemical changes which must occurduring the heating of proteins as such, and be responsible for the nutritional damage.Because of the multiplicity of functional groups and of the many possible reactionswhich may occur during the heating, analytical work is difficult, and very little isknown about what actually happens. Apart from amino acid destructions only thebinding of lysine is well established. It has been suggested that the binding of lysineis caused by a reaction between the -amino group of lysine and carboxylic groups inthe protein and that such cross-linking reduces the nutritional availability of all theessential amino acids (cf. Mecham & Olcott, 1947).

* Paper no. I : B r. J . Nutr. (1969), 3,859.-t Present address : Icelandic Fisheries Laboratories, Reykjavik, Iceland.21 N U T 24


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314 J. BJARNASONND K. J. CARPENTER I970E X P E R I M E N T A L

Test materialsBovine plasma albumin (BPA) and lactalbumin were from the same source as in

our first study (Bjarnason & Carpenter, 1969) and heated samples were prepared inthe same way. The vacuum-dried cod fillets, Cod 23, were similar to those previouslydescribed (Miller, Carpenter & Milner, 1965). The casein was purchased as Casein(Hammersten) (British Drug Houses, Poole, Dorset). The haemoglobin was crystal-lized beef material (Sigma Chemical Co., St Louis, Miss., USA). The egg albumenwas a five times crystallized product (Nutritional Biochemicals Corp., Cleveland,Ohio, USA) and the zein was from the same source.

Analytical proceduresThe moisture, total N and available and residual lysine in samples were deter-mined as in our earlier experiments (Bjarnason & Carpenter, 1969).Free ammonia. Each ampoule, containing about I g protein, was placed in a 1000 ml

polyethylene bottle together with a heavy piece of iron; 10ml 0 .1 M-citric acid and30 ml water were added. The bottle was secured with a screw cap and shaken vigor-ously to break the ampoule and mix the protein thoroughly with the liquid. After 1 0 minthe contents were filtered and made up to 50 ml. The free ammonia was then deter-mined in a sample of the solution according to Hamilton (1960). The unprocessedmaterials were analysed in the same way, except that they were placed dry in thepolyethylene flask.

To tal pri ma ry amide content. This was determined according to Hamilton (1960) onthe dry test materials, after refluxing with 2 N - H C ~or 3-5 or 7.0 h. The average ofthe values obtained after these two intervals was used, and the corresponding valuefor free ammonia was subtracted to obtain the measure of the amide content.

Tot al amino acids. These were determined on acid hydrolysates of the test materialsprepared according to Weidner & Eggum (1966), i.e. hydrolysates were prepared, onehydrolysate in 6 N-HC1, for all amino acids except methionine and cystine, and one in6 N - H C ~fter preliminary oxidation for these two amino acids. Th e amino acids weredetermined on the hydrolysates with a Beckman Amino Acid Analyser 120B (Beck-man Instruments, Inc., Spinco Division, Palo Alto, Calif., USA) by a fully automaticion exchange column procedure described by Spackman, Stein & Moore (1958).Calculations were based on a comparison with a mixture containing all the amino acidsin the concentration of 0.5 ,umoles/ml except for cysteic acid; for this a special calcula-tion technique was followed, as specified in the instruction manual supplied with theinstrument.

When only the determination of total lysine was required, it was carried out on ashort-column system (Roach, Sanderson & Williams, 1967).

N-terminal amino acids. These were determined with 1-fluoro-2,4-dinitrobenzene(FDNB). The methods of Fraenkel-Conrat, Harris & Levy (1955) were followedfor the reaction (procedure I) , hydrolysis of the proteins in 6 N-HCI at 105 for 24 h


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Vol. 24 Changes in heated protein s 315and extraction of the dinitrophenyl (DNP) compounds. The DNP compounds weredissolved in water-free acetic acid and the extinction of the solution was read at 435 nm,a wavelength at which dinitrophenol in acetic acid has negligible extinction. The sumof N-terminal amino acids was expressed as 'DNP-leucine equivalents', i.e. usingthis compound as a reference standard. A correction was made for the compounddi-DNP-lysine. Portions of the acetic acid solution were evaporated in vacuo and theresidues taken up in methanol and transferred quantitatively on to ready-madeKieselgel F 254 plates (Merck AG, Darmstadt, Germany) in a line at the bottom ofthe plates. The chromatograms were developed with benzene-acetic acid-H,O(I :I :I by volume, upper layer used) for direct isolation of leu (+Val), phe, ala andlys (+ yr). The remaining DNP compounds could not be separated with this solventsystem and were re-run for separation in n-butanol-acetic acid-H,O (4: :I byvolume). In this way glu, asp, ileu and ser (+ hr) were isolated. The silica materialcontaining each compound was scraped separately off the plates and the DNP com-pounds were eluted with dilute aqueous NaHCO,. The solutions were filtered, acidifiedwith acetic acid and evaporated. The residues were taken up in glacial acetic acid andthe extinction read at 435 nm. Again, the extinction of DNP-leucine was used as areference. For final identification, the DNP-derivatives were run in three solventsystems, the two already mentioned and chloroform-pyridine-acetic acid (40: 0: byvolume), together with authentic reference samples of each compound.

Free H,S. The ampoules with the heated protein were placed, as for the ammoniadetermination, in a polyethylene bottle together with 20 ml 0.1M-citric acid. Beforebreaking the ampoules, 0,-free N, was led through the flask for 5 min to flush outthe air. Then the ampoule was broken from outside by hammering on the bottle andthe nitrogen stream led for I h through a suspension made by mixing 10 mi 0.1N-Cd(OH), with 2 ml 0.1N-NaOH. The quantitative determination of H,S in thesuspension was then carried out according to Marbach & Doty (1956).Check deter-minations were also made by titration with NazS,O, (Association of Official AgriculturalChemists, 1965, ection 31.016) nd they gave essentially the same results.

Othe r volatile sulphur compounds. The ampoules were placed in a polyethylene tubeconnected with an evacuated flask. The flask had a small rubber balloon connected forpressure equilibration. After breaking the ampoules in the tube and equilibrating thepressure with N,, a sample of gas was taken from the balloon with a syringe and injec-ted into a gas chromatograph (Pye Ltd, Cambridge; dual flame ionization, series 104).Th e column was 5 ft long and contained 100-150mesh Porapak Q. he carrying gaswas argon (45ml/min). The starting temperature was 130', but after 60min wasincreased 5"/min up to 190'. Reference materials were run both separately and mixedwith the gas under study.

RESULTSAmino acids recovered after acid hydrolysis

The destruction of amino acids at high temperatures can be measured by deter-mining the amino acids recovered from hydrolysates of the test materials before andafter they have been heat-damaged. Th e material used for this purpose was crystalline21-2


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316 J. BJARNASONND K . J. CARPENTER I970BPA. It was heated at 115' fo r 27 h t o represent t he conditions of severe processingthat might occur in industry and also at 145' fo r 27 h, .e. under deliberately exag-gerated conditions, so that small trends of losses at I 15' could be confirmed. TheBPA contained 13.9 oH,O in each instance. T h e amino acids were determined afteracid hydrolysis with an amino acid analyser and the results are shown in Table I.

Much the greatest loss occurred with cysteine, 50 yoat I15' and 92yo a t 145'. I ncontrast, methion ine show ed an apparent increase of 14 nd 12 orespectively bu t th eabsolute quantities are so much smaller that the differences, of approximately 0.1g/16 g N, re probably not significant.Table I. Tota l amino acids in bovine plasma albumin (BPA) nd the effect of heating onamino acid content, liberation of ammonia and hydrogen sulphide and total weight

Heated (I I 5", 27 h) Heated (145", 27 h)

Controlk / I O O 9)"

Lysine 12.89Histidine 3'70Arginine 5.82Aspartic acid 10 90Threonine 5.69Serine 3'82Glutamic acid 18.31Proline 4.16Glycine 1.84Alanine 6.12Cyslzt 6.50Valine 6.25Methionine 0.83Isoleucine 2.79Allo-isoleucine -Leucine I 1.98Tyrosine 4.89Phenylalanine 6.46Tryptophan (0.58)f.N recovery in amino acids 15.39Amide-N 0.70Free NH,-N 0'00Total N accounted for I 6.09

(99.3)N accounted for as %of Kjeldahl N in control)Wt changes on heating:Total -Portion accounted for -

Portion accounted for -Unexplained portion -as NH,as H,S

gl100 g"12.413.685.87

10.735'503 6 819.064'576.173'306.1 I0.922.79

12.064.466.38

(0.581115.000.700.23

15'93(98.4)

2'02

-

----

Change (asyo of controlvalue)-4< 3< 3< 3-3-4+ 4+ 1 0+ 1 0< 3- 0< 3+ I4< 3< 3-9< 3

---

- .54- .27-0.17-0.10

Change (a s% of controlvalue)- 5- 7- 2-32- 4< 3+ 9+38+ 14- 2

< 3+ I 2

- 1 1

- 2-< 3+ 51 3--- .08- .04- .47- '57* All values calculated per IOO g ash-free dry matter which contained 16.20g N. In heated materialsweight lost during heating was also taken into account and all values were related back to 100 g of

original ash-free dry matter.t A correction factor of 1.12 was used, bringing thedeterminedvalue for control material to that foundby Stein & Moore (1949).1 Tryptophan was not determ ined; the value of 0.58 (Stein & Moore, 1949) has been used for esti-mating the total recovery of N.


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Vol . 24 Changes in heated proteins 3 I7T h e three dibasic amino acids all show losses, amou nting to I 1-17 % at th e higher

temperature but at I I so only lysine sho ws a loss in excess of 3 %. As a general workingrule, in view of th e known experimental errors in th e system, we are considering onlydifferences th at are a t least 0.4 g/16 g N in absolute am ou nt and representing a relativechange of a t least 3 yo.

Threo nine an d serine both show a high loss at 145' bu t not at th e lower temperature.Aspartic acid does the same, but there is apparently no loss of glutamic acid. N odistinction, of course, can be made between asp artic acid and asparagine, or betweenglutamic acid and glutamine, when analysing hydrolysates of proteins prepared byrefluxing with acid as this causes hydrolysis of the amide groups.

Of the amino acids with a paraffin side-chain, there was a loss only with isoleucine.This is due to racemization, as will be explained below. However, both glycine andalanine show ed significant increases, with a 1 0% increase in th e forme r even at 115'.

Th er e was the same apparent incease of 10 oo in t h e level of proline at each level ofheating. Because of t he g reater inaccuracy of th is determination, an d its show ing notendency to increase at the higher tem perature, no significance is attached to it.

Nearly all the N is accounted for in the material heated at 115' but, after heatingat 145', 7 % has been lost even when amide-N and free NH, have been allowed for.

Table 4,which show s th e results of another ex periment to be considered later, alsoincludes measurem ents of th e destruction of lysine in six different protein materialseach heated at I 15' and 145' for 27 h. T h e accuracy of th e total lysine values for th efive materials other than BPA may b e lower as they were obtained by th e rapid short-column system. Th er e is an average loss of 3yoa t I 15' and of 19yo at 145' in these sixproteins. T h e losses vary considerably from one protein t o another withou t any ap-parent relation w ith th e amino acid compo sition.

RacemixationBPA heated at 145' showed a decrease in isoleucine b ut n ot in th e other am ino acids

with paraffin side-chains (Table I). On the amino acid chromatogram a previouslyunidentified peak also appeared. It was identified as allo-isoleucine (cf. Piez, 1954),

Tab le 2. Contents of isoleucine, allo-isoleucine and lysine (to tal and bound)in vacuum-dried cod Jillets, heated and unheated

Heat-damaged 7Unheated I I ~ " ,4 h 130, 24 h 145', 24 h(a) soleucine (g/16 g N) 4'33 4'27 4'29 3.86(b) Allo-isoleucine (g/16 g N) o t 0.118 0.181 0.49a + b (g/16 g N) 4'33 4'39 4'47 4'35Racemization (yo) < I t( d ) Bound lysine" (g/16 g N) 0.71 2.25 428 5.41

4' 1 11.3- -'7-c) Total lysine (g/16 g N) 8.52* Determined by the procedure of Roach e t a l . (1967).t The hydrolysate showed a small bum p at the point corresponding to allo-isoleucine on the tracebu t it was too small for calculation. A small degree of racemization may have occurred during the acid

hydrolysis of the protein.


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318 J. BJARNASONND J. J. CARPENTER '970a racemized prod uct of L-isoleucine, and was present i n an a mo un t equivalent, withinexperimental error, to the loss of isoleucine.

Examination of the amino acid chromatographs prepared fro m hydrolysates of codfillets heated i n various ways showed com parable results to those obtained with BPA(Table 2). Formation of vola tile compounds

T h e weight loss of proteins du ring heating gives an estimate of th e volatile conden-sation produ cts formed. BPA (13.9 oH,O) was placed in equal am ou nts in a series ofampoules and sealed. After heating some of th e ampoules, they were all opened an d

Table 3. Liberation of ammonia and changes in amide content and FD NB -ava ilablelysine in ji ve proteins as a result of heating(Results are given in m-equiv./16 g N unless stated otherwise)

Analysis beforeheating-

DNB-available Amidelysine groups59'2 46.2Lactalbumin Heated at 63.2 8 1 7Egg albumen 115' for 54 h 43'1 86.20.7 2 1 278.6 55'478.6 55'4

Haemoglobin

ZeinBPA heated at I I 5O for 27 hBPA heated at 145' for 27 h

(1) ( 2 )

1Changes observed as a result of heating

FDNB-availablelysine- 0.8-35'1- 6.3- .7- 6.0-61.2

(3 )Amidecontent

(4)- 3'3- 4'9- 4'5- 1.8- .2+7.2

Liberationof NH,(5 )

24'935'126.018.316.261.8

7Liberation Sum ofof NH, as NH, and% of amide amidecontent groups

(6) (7)56 f11.643 + 10'230 + I I ' 58.5 +6*529 + 10'0

I 1 2 + 69.0FDNB, I-fluoro-2, 4-dinitrobenzene; BPA, bovine plasma albumin.

th e contents dried in a vacuum desiccator over P,O,. T h e difference in weight of theheated an d control material was recorded as weight lost. T h e results are shown inTable I. After heating and drying, some free NH, was still present. It was determinedseparately and the equivalent weight added to the weight lost.

At I I 5 the volatile products formed during heating seem to be mainly NH, andH,S. After heating at 145' these two compounds account for only half of the totalweight loss. T h e H,S corresponded to 22 yoof th e cystine destroyed at I I5' and to 34yoof that destroyed at 145'.

The liberation of volatile sulphur compounds other than H,S has already beenmentioned, but no quantitative determination could be made. Methyl mercaptan,dimethyl sulphide and dimethyl disulphide were detected after heating at 145', withthe use of a gas chromatograph. Thes e compounds too mu st be considered as destruc-tion p roducts of cystine, as there had been n o detectable loss of methion ine.At the higher temperature, i.e. 145', it seems possible that additional volatileproducts suc h as CO, could also have been liberated. T h e evolution of H,O can hardlyhave amounted to m ore than I yo of dry weight.


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Vol. 24 Changes in heated proteins 319Changes in amide groups

The most evident chemical change in BPA heated at 115' is the liberation of NH,.Since the destruction of amino acids, as determined after hydrolysis, is low at thatTable 4.Contents of amide-N, cystine and lysine (total and FDNB-available)in six proteins and the effect of heat on their lysine values

Change in lysine valuesas a result of heating (g/16g N)omposition of unheated materials *r , < ,Lysine (g/16g N) At 115' for 27 h At 145' for 27 hw-c-h--\Cystine' Amide-N" FDNB- FDNB- FDNB-Material (gl16gN) % of total N) available Total available Total available TotalHaemoglobin 0 4'04 8.7 9.1 -1.4 -0.2 -6.2 -1.4Casein 0.22 9.12 7.7 7.8 -1 .7 +0.2 -6.1 -1.6

Cod 23 1.1~ 10.75 8.4 8.7 -2.4 -0.6 -6.6 -2.7Lactalbumin 6 . 1 ~ 8.67' 8.2 8.0 -2.8 -00'2 -6.8 -1.6BPA 6.36 4 76 1 1 . 5 12.7 - 2 . 3 -0 .5 -9.0 - 2 .0FDNB, 1-fluoro-z,4-dinitrobenzene; PA, bovine plasma albumin.' he cystine (not cysteine) values have been taken from the literature for each material, as have theamide-N values for egg albumen and lactalbumin.'Snow (1962); aWallace & Aiyar (1969); SLewis, Snell, Hirschmann & Fraenkel-Conrat (1950);4Connell(1958);Miller, Hartley & Thomas (1965);6Weil& Seibles (1961); OEdsall (1954); Gordon &

Ziegler (1955).

Egg albumen 0 . 5 ~ 6 . ~ 8 ~ 5.9 6.0 -1.2 - '0 -4 .5 - 1 . 3

Decrease in FDNB-available lysine (m-e quiv .ll6g N)Fig. I . The correlation of lysine binding (measured as decrease in FDNB-available lysine)and liberation of ammonia in heated proteins. 0,our materials heated at 115' for 54 h (seeTable I ) ; 0 ,BPA heated at I IS', 27 h; A, PA heated at 145', 27 h. Th e straight line corres-ponds to a I : equivalence. FDNB, 1-fluoro-2,4-dinitrobenzene; PA, bovine plasmaalbumin.


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320 J. B J A R N A S O N AND K. J. CARPENTER I970temp erature, it would seem that th e amide groups of asparagine and glutamine mu stbe its major source. T h e only alternative would be tha t all the N corresponding to th edestroyed cystine (Table I ) was liberated as NH,, bu t thi s seems unlikely.T o s tudy the NH, liberation more closely, six different proteins were heated insealed ampou les un der N, after moisture equilibration over 37 % H,SO, in a vacuumdesiccator. F ree amm onia was the n determ ined and, after drying over P,O, and KOH,th e total amide contents an d FDN B-available lysine were also determined. T h e resultsare shown in Ta ble 3, expressed in m-equiv./16 g N , so as to be relative to th e actualnum ber of th e groups involved, an d to bring o ut the relationship between th e differentchanges.

Arnide content (yoof N) x total lysine ( g / l b g N )Fig. 2. Th e binding of lysine in proteins heated at I I 5" for 27 11 in relation to the product of theinitial concentrations of total lysine and of amide groups in the protein. 0,ncorrected valuesfor sixmaterials from the values set out in Table4; , he samevalueswith the change in F DNB -available Iysine reduced by an amount equimolar with one-quarter of the initial cystine contentof each protein.

By comp aring columns 3 and 5 it can be noted th at th e liberation of NEI, is in fourinstances approximately equivalent to th e binding of lysine an d in th e othe rs is higher ;this is illustrated in Fig. I . I n zein th ere is, of course, negligible lysine available fo rreaction. Comparison of columns 3 and 4 ndicates th at th e lysine binding is generallygreater than the decrease in amide content.

If th e major cause of lysine binding w ere a reaction between t he e-amino gro up oflysine and t he am ide groups of asparagine an d glutamine, a correlation would be expec-ted between th e lysine boun d d uring heating and t he multiplication prod uct of th einitial concentrations of amide groups and total lysine respectively, so long as the


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Vol . 24 Changes in heated proteins 321changes were relatively small. Information suitable for studying th is was obtained fromthe results of another experiment (Table 4) in which six materials had been h eated i nampoules in the usual way, and the values for the milder treatment ( I I 5', 27 h), forwhich a correlation was expected, are set out in t he form of a correlation diagram inFig. 2 . T h e possible significance of th e results will be considered in t he discussion.

Hydrolysis of peptide linkages during heatingA possible chemical change d urin g th e heating of proteins in th e presence of water

is a hydrolytic cleavage of t he peptide chains. I n order to stu dy this, BPA containing13'9% H,O was heated a t I 15' and 145' for 27 h in the usual way. T h e N-terminalamino groups were determined with the D NP-technique, both o n control and heatedBPA. T h e results are shown in T able 5 expressed in nu mb ers of amino grou ps permolecule in th e intact protein, or for th e corresponding weight of the heated proteins.

Table 5. N-ter min al amino acids in control and heated bovine plasma albumin( B P A ) expressed as numbers pe r original molecule*)Heated at Heated at

Intact 115'for 27 h 145'for 27 hAlanineAspartic acid ( + sparagine)Glutamic acid (+glutamine)IsoleucineLeucine (+traces of valine)LysinetPhenylalanineSerinet

0I'00

000000

0.380.29

0.320 1 7

x '00

0

0'21

0'1 I

1'000 3 50.430.090.660.250.420.14

Total 1'00 2.48 ( 2 ' 7 ) f 3'34 (3.4)f* Assuming a molecular weight of 66000 (Edsall, 1954; Thompson, 1954).t Plus traces of an unknown material ( s ee p. 321)1 Th e values in parentheses were obtained by measuring the total extinction of the mixture of DNPamino acids before chromatographic separation.

T h e molecular weight of BPA is ca.66000 (Thompson, 1954;Edsall, 1954)so that onemolecule contains ca . 600 peptide links. After heating at 115' 2.7, N-terminal aminogroups per mole were found. That is an increase of 1.7 which corresponds to about0.3 yo of the peptide links and must be considered as very low. N-terminal histidinean d arginine were not determined bu t their DN P-derivatives appear in th e blank inth e FDN B-available lysine determination, t he value for which was very low. At 145'th e total num ber of N-terminal am ino groups per molecule was 3-4 which is not mu chhigher. It is interesting to note th at th e N-terminal aspartic acid has decreased at thesame time as all the others have increased. Glycine and cystine were not found asN-terminal amino acids; this could be du e to the known sensitivity of their D N Pderivatives to destruction du ring hydrolysis (Porter & Sanger, 1948). T h e unidentifiedtraces in th e lysine an d serine values are probably the D N P derivatives of tyrosine andthreonine respectively. A lmost all the amino acids seem to app ear as N-terminal afterth e protein has been heated in th e presence of water. How ever, the degree of hydrolysis


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322 J. BJARNASONND K. J. CARPENTER I970is low and would correspond to an increase of only about 0 . 0 5 ~ ~n dry weight dueto the postulated reactions with water.

Mo del reactions involving amidesT o investigate whether s-amino lysine groups in proteins are acylated by heating

with amides, two pure proteins were heated with acetamide and N-acetyl-glutaminerespectively. As controls, we repeated the heat-processing in the presence of ammoniumacetate and N-acetyl-glutamic acid monosodium salt respectively. All additives werethoroughly mixed with the proteins in a mortar and equilibrated as usual over 37%H,S04 before heating. The results are shown in Table 6.

Table 6. Eflects of heat on FDN B-ava ilabl e lysine in haemoglobin and B P A in thepresence of amides or ammonium salts

FDNB-available lysineTotal amide (g/roo g protein) Boundcontent before 7-ysinetheating* (m-equiv./ In Change with (g/Ioo gIOO g material) material heating protein)Haemoglobin : - -nheated control 46.2 8.50 - .78eatedf alone 46.2 5'72 -3.83eatedf with 14.5yo ammonium acetate 39'4 4'67Heatedf with I I ' 5 % acetamide 236 I 5 2 - -98Unheated control 55'4 I I .62 - 0.8 I---BPA :Heated5 alone 55'4 9'3 5 - .27 2'47Heated with 23.3 yo N-acetyl-L-glutamic 42'5 8.38 - '24 3.08

Heated5 with 21.4 % N-acetyl-L-glutamine I j6.5 6.75 -4.87 4.16acid monosodium saltFDNB, ~-fluoro-z,+dinitrobenzene;BPA, bovine plasma albumin.* Only primary amides measured. Th e ammonium salt was added in each case at a level equivalent to thatt Determined as 'unreacted lysine' after FDN B treatment and acid-hydrolysis (Roach e t a l . 1967).1 115' for 54 h.4 115' for 27 h.

of the amide, added to the same protein.

Haemoglobin heated with acetamide shows a much greater Iysine binding, i.e. de-crease in FDNB-available lysine, than does haemoglobin heated alone. The ratio ofthe initial rates of reactions must, of course, have been even higher than the finallysine binding ratio, 6.98/2.78 = 2.51, ince the reaction rate decreases as the levelof available lysine falls. Only 18% of the original FDNB-available lysine remains afterheating with acetamide. Haemoglobin heated with ammonium acetate shows a littlehigher binding than when heated alone, but the effect is much less than with acetamide.BPA heated with N-acetyl-L-glutamine behaves similarly. Here the final lysinebinding ratio is 2-14, .e. slightly lower than for haemoglobin, but the ratio of totalamide with and without the supplement is 2.8 which is also lower than the correspond-ing value of 5-1 in the haemoglobin experiment. Again, with BPA the control additiveN-acetyl-L-glutamic acid monosodium salt caused a higher binding of lysine thanwhen BPA was heated alone. The action of the two control additives will be consideredin the discussion.


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Vol. 24 Changes in heated proteins 323From these experiments it seems clear that a reaction between the s-amino groupof lysine in proteins and primary amide groups is possible.

D I S C U S S I O NThe losses of 50% of the cystine and 4 yo of the lysine from heating BPA at I I 5'for 27 h, as seen in Table I , were in good agreement with the results of Miller, Hartley

& Thomas (1965)' who found a decrease of 60% in cystine and 6% in lysine in vacuum-dried cod heated at 116' for 27 h. Other authors (reviewed by Miller, Hartley &Thomas, 1965) report similar findings; also that the presence of carbohydrates aggra-vates the loss of lysine (apart from its binding) and causes losses of arginine as well,even at temperatures around 100'. Heating BPA at 145' has caused some destructionof a large number of amino acids and again similar observations have been made withvacuum-dried cod (J . Bjarnason, unpublished results).

Apparently at temperatures only a little above IOO', that is at practical dryingtemperatures, only cystine seems to suffer important losses in carbohydrate-freeproteins. It is known that hot water releases some H,S from wool protein and that thisH,S comes from the disulphide bridges of cystine (Schoberl, 1941). The followingmechanism of a reaction between water and disulphide groups under similar con-ditions has been postulated (Schoberl & Eck, 1936):

' A ' 'B'R-CHZ-S-S-CHZ-R +HZO -+ R-CHZ-SH +HOS-CHZ-R ( I )

HOS-CHZ-R -+ HZS+OCH-R (2)It seems probable that such a mechanism is at least partly responsible for the loss

of cystine that occurs during the heating of proteins in the absence of oxygen. Afterheating BPA at I IS', H,S corresponding to about 20% of the lost cys/z was recovered,but about 30yoafter heating at 145'. Similarly, Schoberl(1941) states that the recoveryof H,S after heating wool in water is not a direct measure of the disulphide cleavageas some of the H,S will not be found and that the loss of cystine is a better indication.After heating BPA at 115' only small quantities of cystine were found after acidhydrolysis, but 51yoof the original content was found after oxidation, as cysteic acid.This shows that, after heating at I I ~ ' , the cys/z is mainly present as cysteine. Thecontrol material gave almost all of the cys/2 content as cystine. The 49 yo loss of thecys/z content in this case and similar losses reported by others for a variety of materialsheated above 100' (cf. Miller, Hartley & Thomas, 1965) are perhaps significantevidence of an easy destruction of one half of the c y s / ~ontent according to equation

The second stage (2) of the equation is not very clear. In some instances aldehydeshave been reported as products (Schoberl & Eck, 1936) but, depending on the pH,dismutation of product B may also take place (Schoberl, 1933).In BPA heated at 115' the sum of free and amide-bound ammonia has increasedfrom 0.785 g to 0.925 g N/16 g N. This can only be explained by a destruction ofamino acids with the transformation of a-amino-N into free or amide-bound am-monia; it cannot be explained by losses in arginine, histidine or lysine. The N must

(1).


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324 J. BJARNASONND K. J. CARPENTER I970therefore come from cystine, as there are no detectable losses in other amino acidsexcept lysine and possibly tyrosine and it means that the peptide chain must havebeen ruptured. It may be that the sulphur of cystine has also been liberated to someextent through a p-elimination to form a dehydroalanine in the peptide chain, whichthen subsequently rearranges and easily hydrolyses to form an amide group and apyruvic acid derivative (Patchornik, Sokolovsky & Sadeh, 1961)

kI

RSHI

Step a is the most co mm on sid e reaction of cysteine derivatives in pe ptide chem istry,even at low basicities (Schroder & Liibke, 1965b). A t neutral pH and elevatedtemperatures, a similar mechanism can be expected when R is -S- (a cystinederivative).T h e work of Mecham & Olcott (1947) is interesting in this connexion. Th ey fou ndthat when they heated proteins of low amide content but relatively high cystineconten t (i.e. hoof and egg white) in a water-free suspension in hydrocarbon the am idecontent increased, but that when proteins with higher amide contents and lowercystine content (casein, gluten, zein, soya-bean globulin) were heated in th e sam e way,there was a decrease in their amide content. T he se results are not d irectly com parablewith ours, since very little water was present, bu t they provide independ ent evidencethat an increase in amide can take place an d a furth er suggestion that this is to someextent related to a breakdown of cystine.


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Vol. 24 Changes in heated proteins 325T h e liberation of methyl mercaptan, dimethyl sulphide and dimethyl disu lphid eat 145' indicates also another destruction mechanism of cystine involving th e ru ptu reof C-C bond s at tha t temperature. T he re was apparently no loss of methionine , th eonly other source of sulphur.T h e loss of lysine recoverable from acid-hydrolysates as a result of hea ting B PA a t115' cannot be explained by a reaction of the e-amino group with the amide groupsof asparagine and glutamine. T hi s would rat her mean a protection. Ta bl e 4 shows thelosses of lysine in six proteins. T h e two essentially cystine-free proteins, haem oglobinan d casein, seem to show lower lysine losses tha n th e others at I 15' though not at 145'.

It has been demonstrated that alkaline treatment of proteins followed by acid-hydrolys is can yield N-~-(~~-~-amino-2-carboxyethyl)-~-lysineBohak, 1964;Ziegler,Melchert & Lurken, 1967). It is postulated that this is the result of a break-dow n of cystine residues to give dehydroalanine residues a nd th en th e addition of a nE-NH, group of a lysine residue across the double bond. N o peak corresponding toth e lysine-alanine comp ound has been detected in hydrolysates of our heated samples ;nor would it be expected in material that has been heated at a neutral pH.

In almost all other possible mechanisms for cystine degradation, carbonyl groupsappear as products. T h e lysine losses durin g heating of proteins at 100' or above mayvery well be the result of a Maillard-type reaction between the -amino group andcarbonyl compound s such as may result from the destruction of cystine. T h e appar-ently low degree of correlation between th e lysine destruction an d cystine content in thecystine-containing proteins (Table 4) may be the result of different degrees of re-arrang em ent of Schiff's bases. Some Schiff's bases an d other amino-carbony l produ ctsare more stable than others, depending on m any factors, and yield the amine com-ponent intact on acid hydrolysis (Bujard, Handwerck & Mauron, 1967).At 145' the dest ruc tion of c y s / ~nd lysine in BPA has increased considerably. Th er eis a considerable destruction of histidine, arginine, serine, threonine and aspartic acidas well. T h e increase in free and am ide-bound ammonia is 0.972 g N/16 g N. T h i sexceeds the amount of NH, possibly liberated by cystine deterioration (maximal0.699 g N/16 g N ) so tha t other amino acids have obviously contributed. Serine andthreonine are known to liberate amm onia slowly on acid hydrolysis (Smy th, Stein &Moore, 1962), probably over a p-elimination of water (classical acid-catalyseddegradation of a-a mi no alcohols) followed by a similar rearrangement and hydrolysis totha t set ou t in reaction b. Certainly th e BPA had a very weak acidic pH , b ut a n elevatedtemperature might have th e same effect. Th e imidazole nucleus of histidine an d th eguanidyl group of arginine are potential sources of ammonia as well.

It is interesting to note that at 145' the re is a small increase in glycine a nd a con-siderable increase in alanine. It is not easy to see how any other amino acid exceptaspartic acid could b e transformed into alanine. It is known tha t asparagine and aspar-tic acid in peptide chains form a-amino-succinimide derivatives du ring heat treatm entin boiling water (Riniker, Brunner & Schwyzer, 1962; Schroder & Lubke , 1 9 6 5 ~ ) .Su ch products may possibly decarboxylate to some extent to give alanine; the loss inaspartic acid is in line with this. It is interesting tha t irradiation has also been fo un dcommonly to increase th e alanine content of proteins (Ro ubal & Tappel , 1966).


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326 J. BJARNASONND K. J. CARPENTER '970Th e total N recovery was almost IO O yoboth for intact BPA and for BPA heated at

115' (Table I) . After heating at 145', 6.5 yoof the N remained unaccounted for; thiswill be included in other nitrogenous compounds than amino acids or free and amide-bound ammonia, as a result of more complex deterioration.The changes in amino acid composition during heating of proteins, which havealready been discussed, are not of much nutritional significance unless the destructionof cystine were to make the sulphur amino acids limiting. Normally, other changes inthe proteins affecting availability reduce the nutritive value much more, overriding therelatively small changes in amino acid composition (cf. Miller, Hartley & Thomas,1965). As the main nutritional damage is not detected by amino acid analyses on acid-hydrolysates, it is obvious that the changes responsible are largely reversed on acidhydrolysis, that is to say, they are a result of condensation reactions. The binding oflysine during the heating of carbohydrate-free proteins, which has been known forsome time (Carpenter et al . 1957, 1962; Lea, Parr & Carpenter, 1960) must be of thisnature, as chemically bound lysine can be liberated on hydrolysis, and is measured asresidual lysine in the procedure of Roach et al. (1967).

It has sometimes been suggested that the binding of lysine in carbohydrate-freeproteins during heating is due to the formation of unnatural 'amide' bonds betweenthe s-amino group of lysine and carboxylic groups in the proteins. Certainly thesewould be reactions that could not be detected by total amino acid analyses. Our resultssuggest the formation of the same products but by reaction between the s-amino groupof lysine and the amide groups of asparagine ( n = I ) or glutamine ( n = 2) , with thereacting units either in the same peptide chain or in neighbouring ones.

0II-NH, ,C-

CHI0ll-NH /C-'CH

The following points, both from our results and from the literature are in favour ofthis.

( I ) As seen in Table I , ammonia is the main volatile product formed during theheating of BPA at 115'. There is reason to believe that the liberation of water as a


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Vol. 24 Changes in heated proteins 327result of condensation between e-amino groups of lysine and carboxylic groups is lowif any, unless there is some unexpected chemical uptake of water, as the recovery ofweight loss in NH, and H,S is about 82% (Table 2).

A water uptake according to reaction b above would only amount to about 0.1 % ofthe dry weight and, against this, the carbonyl compounds formed could lose theadded water in condensation reactions. The water uptake as a result of hydrolyticcleavage of peptide bonds is extremely low, as has already been discussed.

(2) Liberation of NH, in five proteins heated at 115 for 54 h is higher than orequivalent to the lysine binding (Table 3). The liberation is not a direct function of theamide content alone, as is seen in column 7, which indicates that it is the result of someinteraction though, obviously, small amounts of NH, may also be liberated by hydroly-sis of the amide groups.

Zein which has far the highest amide content does show a small liberation of NH,,even though it has almost negligible lysine content. This represents a deviation fromthe general correlation between the lysine binding and the liberation of NH, (Fig. I).Four of the proteins show lower amide decreases than lysine binding. Here again zeinis an exception, but the relative change in the amide content of this material is low.

The decrease in the amide content as a result of some specific reaction is complica-ted by the fact that, mainly at higher temperatures, there is another factor causing theformation of amide groups. This is seen from BPA heated at 145 and from the resultsof Mecham & Olcott (1947). Column 7 in Table 3 gives an idea how much this couldamount to at 115.

(3) Table 4 shows that the lysine binding in six proteins seems to be dependent onthe amide content of the protein as well as on the lysine content. This suggests that abimolecular reaction is involved and one would expect the rate of reaction to beroughly proportional to the product of the concentrations of each reactant. A correla-tion diagram showing the available information has been plotted in Fig. 2.

If, as has been argued above, the destruction products of cystine also play a part inthe binding of lysine through their carbonyl groups, one would have to allow for thisbefore estimating the extent of the reaction between amide groups and lysine. InFig. 2 a deduction has been made for this on the arbitrary assumption that one-half ofthe cystine has been destroyed and that one-half again of the destroyed molecules haveyielded a carbonyl group which has combined with lysine. Since the molecular weightsof lysine and cystine are in the ratio 146 : 240, the change in FDNB-lysine (g/16 g N)has been reduced by 0.14 x cystine content (g/16 g N). I t is seen that these adjustedvalues do show a closer correlation with the product of initial amide content andtotal lysine content .(4) Model experiments on haemoglobin and BPA, heated with acetamide andN-acetyl-glutamide respectively, showed that a reaction between amide groups andthe e-amino group of lysine in proteins can occur under the conditions of our heatingexperiments.The same proteins heated with ammonium acetate or N-acetyl-glutamic acid mono-sodium salt, also showed a higher lysine binding than the proteins heated alone, butthe effect was much less than when the amides were added. Perhaps these control


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328 J. BJARNASON AND K. J. CARPENTER I970materials were poorly chosen as ammonium acetate contributes ammonia, whichcould transform cyclic aspartic and glutamic acid residues into amides and so indirectlyincrease the amide content of the protein. N-acetyl-glutamic acid monosodium saltalso contains a secondary amide group, which could possibly acetylate the -aminogroup of lysine to some extent.

Reaction between carboxylic groups and the eamino group of lysine is not verylikely on thermodynamic grounds. High temperatures and a removal of water areneeded to bring about an appreciable condensation between these groups.

(5) Ford & Salter (1966) found that aspartic and glutamic acids in cod fillet heatedat 135' were poorly released by enzymic digestion as compared with amino acids otherthan lysine and cystine. Erbersdobler, Dummer & Zucker (1969) studied the absorp-tion of amino acids from heated casein in the portal blood of growing rats. They foundglutamine and asparagine to be the acids most reduced after lysine in availability inprotein that had been heated at 105'. Our results suggest that simple hydrolysis duringheating of the amide groups of these amino acids is low. Thus, the findings of Erbers-dobler et al. (1969) seem to indicate a specific reaction involving these two aminoacids.(6) e(y-Glutamy1)-lysine has been isolated from enzymatic hydrolysates of cross-linked human fibrins (Pisano, Finlayson & Peyton, 1968; MatadC & Loewy, 1968) butwas not found in the intact fibrin. This cross-linking reaction was catalysed by theenzyme plasma transglutaminase, i.e. it involved glutamine rather than glutamic acid.It is rather unlikely that this enzyme enables the reaction to proceed through an energysource carried by the enzyme. This emphasizes that a reaction between the amidegroups and the s-amino group of lysine in proteins is at a thermodynamic advantage,even at low temperatures.The nutritional significance of such cross-linkages will be discussed in a furtherpaper.

We are grateful to Mr G. Arnesen, who carried out the amino acid analyses on theBPA and to Dr V. H. Booth, who determined the total and residual lysine valuesreferred to in Tables 4 nd 6. We are also grateful to Dr P. Swaboda for supplying uswith reference materials for the gas chromatographic studies. One of us (J. B.) wasin receipt of a Broodbank Fellowship from the University of Cambridge.

REFERENCESAssociation of Official Agricultural Chemists (I965). Oficial Methods of Analysis, I 0th ed. Washington,Bjarnason, J. & Carpenter, K. J. (1969). Br. J. N u t r . 23,859.Bohak, Z. (1964).J. biol. Chem. 239, 878.Bujard, E.,Handwerck, V. & Mauron, J. (1967).J. Sci . Fd Agric . , 18, 52.Carpenter, K. J., Ellinger, G. M., Munro, M. I. & Rolfc, E. J. (1957). Br. J. N u t r . 11, 162.Carpenter, K. ., Morgan, C. B., Lea, C. H. & Parr, L. J. (1962). Br. J. N u t r . 16, 451.Connell, J. J. (1958). Conference on Fundamental Aspects of the Dehydration of Foodstuffs, Aberdeen,Edsall, J. T. (1954).J . Polymer Sci. 12, 53.Erbersdobler, H., Dummer, H. & Zucker, H. (1969). Z . Tierphysiol. Tierernuhr. Futtermittelk. 24, .

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Vol. 24 Changes in heated proteins 329Ford, J. E. & Salter, D. N. (1966). Br. J. Nutr. 20, 843.Fraenkel-Conrat, H., Harris, J . I. & Levy, A. L. (1955). Meth. biochem. Analysis 2, 359.Gordon, W. G. & Ziegler, J. (1955). Archs Biochem. Biophys. 57, 80.Hamilton, L. D. G. (1960). In A Laboratory Manual of Analytical Methods f o r Protein Chemistry.Vol. 2, p. 72 [P. Alexander and R. J. Block, editors]. Oxford, London, New York and Paris: Per-gamon Press.Lea, C. H., Parr, L. J. & Carpenter, K. J. (1960). Br. J. Nutr. 14, 1.Lewis, J.C., Snell, N. S., Hirschmann, D. J. & Fraenkel-Conrat, H. (1950).J. bid. Chem. 186, 3 .Marbach, E. P., & Doty, D. M. (1956).J. agric. Fd Chem.4, 81.MataEiC, S.& Loewy, A. C. (1968). Biochem. biophys. Res. Comm.30, 56.Mecham, D. . & Olcott, H. S. (1947). Ind. Engng Chem. 39, 023.Miller, E.L., Carpenter, K. J. & Milner, C. K. (1965). Br. J. Nutr. 19, 547.Miller, E. L., Hartley, A. W. & Thomas, D. C. (1965). Br. J. Nutr. 19, 65.Patchornik, A,, Sokolovsky, M. & Sadeh, T. (1961).Proc. int. Congr. Biochem. v. Moscow Vol. 9,Piez, K. A. (1954).J. bid. Chem. 207, 77.Pisano, J. J. , Finlayson, J. S. & Peyton, M. P. (1968). Science, N . Y. 160, 92.Porter, R. R. & Sanger, F. (1948). Biochem.J.4,87.Riniker, B ., Brunner, H. & Schwyzer, R. (1962). Angew. Chem. 74,469.Roach, A. G., Sanderson, P. & Williams, D. R. (1967). J. Sci. Fd Agric. IS, 74.Roubal, W. T. & Tappel, A. L. (1966). Archs Biochem. Biophys. 113,.Schoberl, A. (1933).Justus Liebigs Annln Chem. 507, I I I .Schoberl, A. (1941). Chem. Ber. 74B, 1225.Schoberl, A. & Eck, H. (1936).Justus Liebigs Annln Chem. 522, 97.Schroder, W . & Liibke, K. (1965~) .The Peptides. Vol. I, p. 201 . New York and London: AcademicSchroder, W. & Lubke, K. (1965b). The Peptides. Vol. I , p. 227. New York and London: AcademicSmyth, D. G., Stein, W. H. & Moore, S. (1962).J. biol. Chem.237,1845.Snow, N. S. (1962). Biochem.J. 84, 60.Spackman, D.H., Stein, W. H. & Moore, S. (1958). Analyt. Chem.30, 190.Stein, W. H. & Moore, S. (1949).J. biol. Chem. 178, 9.Thompson, E.0 . P. (1954). J. biol. Chem. 208, 565.Wallace, G. M. & Aiyar, K. R. (1969).J. Dairy Res. 36, 5.Weidner, K. & Eggum, B. 0. (1966).Acta agric. scand. 16, 1 5 .Weil, L. & Seibles, T. S. (1961).Archs Biochem. Biophys. 93, 93.Ziegler, K.,Melchert, I. & Lurken, C. (1967).Nature, Lond. 214, 04.

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