Acylation of Fish Protein:Effect of ReactionConditions on Products

KANG-HO LEE, HERMAN S. GRONINGER,and JOHN SPINELLI

this includes the epsilon-amino groupof lysine, the sulfhydryl group of cys­teine, the phenolic group of tyrosine,and the hydroxyl groups of serine andthreonine when fish myofibrillar pro­tein is acylated under various condi­tions. The inhibitory effect of sulfiteon the acylation reaction was alsoevaluated.

Introduction

Functionality is the attribute of afood material that gives the desiredproperties to the food system in whichit has been incorporated. For exam­ple, it is the emulsion-stabilizing func­tionality of egg yolk that makes possi­ble the fat-in-water emulsion that isthe basis of mayonnaise. It has beenshown that the functional propertiesof fish muscle proteins can be im­proved by chemically modifying theseproteins (Groninger and Miller, 1975;Miller and Groninger, 1976). Modi­fication through the acylation of fishmyofibrillar protein has been de­scribed by Groninger (1973) and Chenet al. (1975). Acylation involves thechemical bonding of a smallmolecule, in most instances acetyl orsuccinyl, to the protein which resultsin an alteration of the electrical chargeon the protein.

Recent studies of the chemical andnutritional properties of acylated fishprotein (Groninger and Miller, 1979)included determination of theirnutrititive value and biological utiliza­tion relative to acylation reaction con­ditions. Rat feeding tests showedclearly that acylation resulted in

The authors are with the Utilization ResearchDivision, Northwest and Alaska FisheriesCenter, National Marine Fisheries Service,NOAA, 2725 Montlake Blvd. East, Seattle, WA98112. Kang-Ho Lee was supported by fundsfrom the Ministry of Education, Republic ofKorea, while working as a visiting scientist at theUtilization Research Division. His present ad­dress is Department of Food Science andTechnology, National Fisheries University ofBusan, 599-1 Daeyeon-Dong, Nam-Gu, Busan601-DI, Korea.

14

decreased nutritional value of themodified protein as measured by theprotein efficiency ratio and the feedefficiency. A modified protein withimproved functional properties andreasonably good nutritional value wasproduced, however, at low tomoderate levels of acylation. Such aproduct would be desirable for foodapplications in which the modifiedprotein additives might be a signifi­cant part of the protein intake.Therefore commercial applications ofthe process will require selection andcontrol of reaction conditions to pro­vide modified protein with the neededfunctional properties as well asreasonably good nutritive value. Forthis purpose, detailed knowledge ofacylation reaction conditions and theeffects on products is important.

Although the major target of theacylation reaction is the amino groupof the amino acid lysine (Grant-Greenand Friedberg, 1970), minor reactionsoccur to form secondary productswith cysteine (Habeeb et al., 1958),tyrosine (Riordan and Vallee, 1963),and with serine and/or threonine(Gounaris and Perlmann, 1967). Inmost reported work on the acylationof proteins for food purposes, littleattention has been given to the forma­tion of these secondary products.Since these secondary products maybe important from the point of viewof nutrition, food safety, and thefunctionality of the modified protein,work on secondary products was in­itiated.

The objective of this work was todetermine the relative reactivity of thevarious sites on the fish protein thatmight involve the acylation reagent;

Methods

The various methods used in thiswork are described as follows.

Preparation of Myofibrillar Proteins

The procedure for the preparationof myofibrillar proteins was based onthe method described by Groninger(1973). Fresh or defrosted rockfish(Sebastes spp.) fillets were com­minuted by passing through a foodgrinder with a 0.2 cm orifice plate.The comminuted fish muscle wassuspended three times in a large excessvolume of cold O. I M NaCl to removea large proportion of water-solubleproteins, minerals, and other extrac­tives. The supernatant was removedby centrifugation for 10 minutes at8,000 g, O°c. To remove additionalsoluble protein, the muscle proteinwas blended for 20 seconds in a high­speed blender with about 3 volumesof 0.1 M NaCI at O°C and centrifugedfor 10 minutes at 8,000 g, O°c.Blending and subsequent centrifuga­tion was also repeated three times.The myofibrillar proteins weresolubilized by blending for 30 secondsin 0.6 M NaCl at O°c. Connectivetissue and nonsolubilized myofibrillarprotein were removed by centrifuga­tion for 15 minutes at 13,000 g, O°C.The typical protein preparation con­tained 30 to 32 mg protein/g.

Acylation of Fish Protein

Salt-solubilized myofibrillar pro­tein, at a concentration of approx­imately 30 mg/g, was acetylated byreacting with acetic anhydride under aconstant agitation in an ice bath atdifferent pH's and with or withoutthe addition of sodium sulfite. Aceticanhydride was added dropwise by amicroburette in a constant rate of ad­dition (0.5 g/minute). Simultaneous-

Marine Fisheries Review

Recovery at pH

Table 1.-Deacetylallon recovery of O· and $·acetylcompounds with hydroxylamine at pH 12.5 and 7.5.

a· and S·mixture 2.63 2.55 97 1.41 54Same amount of

a- and S·mixture

S-mixture 3.00 3.05 102 2.78 931 mM S·acetyl·

thiocholine1 mM S·acetylmer·

captosuccinate1 mM N,S·diacetyl­

cysteine

Analysis of O-acyl Tyrosine

A spectral determination methodfor tyrosine was adapted to estimateacyl O-tyrosine after deacylation withhydroxylamine. This test is based onthe shift of absorption maximumfrom 263 to 278 nrn and an increase inmolar absorptivity when O-tyrosineresidues of protein are deacylated.

In practice, solubilized fishmyofibrillar protein was found tobe too turbid for direct spec­trophotometric analysis withoutclarification by proteolytic treatmentprior to deacylation. This was ac­complished by incubating 10 ml ofsolubilized protein suspension in bar­bital buffer (pH 7.5) with 1 ml oftrypsin-bromelain mixture (50 + 100rng/4O ml H20 for 30 to 60 minutes atroom temperature. If necessary, theprotein solution was incubated for anadditional 60 minutes at 30°C in ashaker bath. The mixture wasclarified by centrifugation for 15minutes at 18,000 g, o°c. Deacylationof O-acyl tyrosine in 1.0 ml ofenzyme-treated protein solution wasaccomplished with 2 ml of 1 Mhydroxylamine reagent (pH 7.5) for10 minutes at room temperature. Thevolume was adjusted to 5 ml withwater, and the absorbance wasmeasured at 278 nrn. The increase inabsorbance at 278 nm between thenative and acylated proteins, Em =

1,100 per mole, was used to determinethe amount of O-acyl tyrosine inacylated fish protein.

Analysis of S-acyl Cysteine

Sulfhydryl was estimated using

temperature. The reaction was stop­ped by addition of 1.0 ml of concen­trated HCl diluted at 1:2 (vIv) andsubsequently 1.0 ml of 1percent ferricchloride in 0.1 NHCl was added. Theprotein precipitate formed was remov­ed by ftItration, and the absorbanceof the supernatant was measured by540 nrn 20 minutes after the additionof ferric cWoride. Quantitation in allcases was based on standard curvesusing acetylcholine and monomethyl­succinate.

7.5

0.25 11

--(mM) %

Con-centra- 12.5

Test tionmixture' (mM) (mM) %

a·mixture 2.25 2.21 981 mM a·acetyl·

hydroxyprol ine0.25 mM a·acetyl·

tyrosine1 mM N,a·diacetyl­

serine

amount of acyl groups on secondaryresidues including tyrosyl, sulf­hydryl, and hydroxyl; and the valuesmeasured at pH 7.5 are the sum oftyrosyl and sulfhydryl residues. Thedeacylation values measured at pH7.5 were very similar to those valuesof O-acyl tyrosine and S-acyl cysteine,which were measured individually bythe methods of Riordan et al. (1965)and Habeeb (1972). The amounts ofacylated O-hydroxyl residues as thetotal of O-serine and O-threoninewere simply computed by subtractingthe amount of O-acyl tyrosine plusS-acyl cysteine from the total deacyla­tion value measured at pH 12.5.

Percent acylation given in the datawas calculated as the percent ratio ofeach secondary residue in mg/100 gprotein based on the contents of cor­responding amino acids from the dataof amino acid composition ofrockfish protein by Groninger andMiller (1979).

The hydroxylamine deacylationreagent consisted of 10 ml of 2 MNH20H and 10 ml of 3.5 N NaOHfor the test at pH 12.5; a mixture of 10ml 2 M NH20H and an appropriateamount of 3.5 NNaOH for the test atpH 7.5. In general procedure fordeacylation, 2 ml of 1 M hydrox­ylamine reagent was added to 1.0 mlaliquots of protein solution andreacted for 10 minutes at room

Iy, 2 N NaOH was added to provide apH stat-like condition during the reac­tion period, which normally took40-60 minutes in a 600 ml of totalreaction volume.

Succinylation was carried out usinga procedure similar to the acetylationprocedure except that incrementalamounts of solid succinic anhydridewere added.

Protein Content

Protein content was determined bythe method of Lowry et al. (1951).

Extent of Acylation

The extent of acylation of proteinwas measured by the method ofKakade and Liener (1969) andcalculated as percent acylation basedon the amounts of free amino groupsin the acylated and the native samples.

Acylhydroxamates

To estimate the amount of acylatedsecondary side groups in protein,acylhydroxamates were determinedby Hestrin' s method (1949).Acylhydroxamates are formed bydeacetylation of acylated residueswith alkaline hydroxylamine andsubsequently the ferric acylhydrox­amate complex is formed which hasan absorption maximum of 540 mm.

In the preliminary work withacetylated compounds, it was foundthat deacetylation with hydrox­ylamine occurred selectively on pro­tein residues under conditions of con­trolled pH. As shown in Table 1, allof the acetyl compounds were readilydeacetylated at pH 12.5 while S­mixture and O-acetyltyrosine werereacted at pH 7.5, although therecovery was slightly reduced.O-acetyltyrosyl derivatives were readi­ly deacetylated when acetylated pro­teins were tested as described by Rior­dan and Vallee (1972). Furthermore,an estimation of cysteine could bemade in the presence of O-acetyl­tyrosine when deacylation was per­formed at pH 6.5 to 6.0. However,this was not satisfactory for a quan­titative test because of variability.

Therefore, the deacylation valuesmeasured at pH 12.5 are the total

March 1981, 43(3) 15

Table 2.-Recovery and optimum reaction conditionsfor deacylatlo~ 01 N,S·dlacetylcystelne and subse·~ent quanlltatlon with S,S'·dlthlobls·(2·nltrobenzolcacid).

appeared to cause fading of the color,the absorbance was read withinseveral seconds after the addition ofthe DTNB reagent.

Results

For the purpose of simplification,the results are presented in the follow­ing six sections.

Acetylation of Amino Group

Salt-solubilized fish myofibrillarproteins were acetylated with aceticanhydride at three different pH's (9.5,7.5, and 6.5), and at various levels ofacetylating agent at oce. The extentof acetylation was expressed in termsof percentage of reactive lysine resi­dues with 2,4,6-trinitrobenzenesulfon­ic acid reagent. As shown in Figure 1,the rate of reaction was affected byboth pH and concentration ofreagent. The reaction proceededmuch faster at pH 9.5 than at lower

Ellman's reagent and the general pro­cedure of Habeeb (1972); however, itwas adapted for use on modified fishprotein. In this modified procedure,acylated sulfhydryl groups weredeacylated with hydroxylamine priorto reaction with Ellman's reagent, andthe difference in absorbance at 412nm between the control and modifiedprotein was measured. A result of thequantitive determination of N,S-di­acetyicysteine through the reactionwith 5,5-dithiobis-(nitrobenzoic acid)(DTNB) reagent is shown in Table 2.At least 2 ml of 1 M hydroxylaminefor deacetylation and 1.0 ml of 2 mMDTNB reagent were required todevelop a quantitive color reaction.The concentration of the sample wasshown to be a critical factor, and theabsorbance of the range of 0.4-0.7was used for best recovery.

For deacylation, 1 ml of 2 percentsodium dodecyl sulfate phosphatebuffer solution (pH 8.0) with 0.2 mlof 0.2 M EDTA and 2 ml of 1 Mhydroxylamine (pH 7.5) were addedto 1 ml of enzyme-treated proteinsolution. After deacylating for 10minutes at ambient temperature, 1 mlof 2 mM DTNB was added, thevolume was made up to 5 ml withwater, and the absorbance wasmeasured at 412 nm. A molar absorp­tivity of 13,000 was used for thecalculation of the amount of S-acylsulfhydryl. Because hydroxylamine

N,S·diacetyl·cysteine(0.2 mM)

(ml)

1.01.01.01.01.01.0

Deacylationwith 1 M

hydroxylamine(ml)

2.02.02.01.01.01.0

2 mMDTNS

reagent(ml)

1.00.50.21.00.50.2

Recovery(per­cent)

995538483427

pH's. The reaction was almost com­plete even under relatively mild condi­tions with 30 to 50 mM anhydride(ratio to protein, 1:8 to 1:5) at eachpH until the proteins were 80-90 per­cent acetylated. At 40 mM anhydrideconcentration, acetylation was about90 percent complete at pH 9.5,whereas only 75 and 65 percent werecomplete at pH 7.5 and 6.5, respec­tively. This shows that differentamounts of anhydride were requiredto provide the same degree of acetyla­tion at different pH's. For example,approximately 30, 45, and 55 mM ofanhydride were added to yield 80 per­cent acetylation at pH 9.5, 7.5, and6.5, respectively. With amounts ofacetic anhydride in excess of 80 mM(ratio to protein, 1:4), the reactionwas completed rapidly, leading toover 90 percent acetylation at eachpH.

The effect of the rate of anhydrideaddition was evaluated; however,significant differences in overall reac­tion rates were not observed when 0.5to 1.0 g/minute anhydride was added.In all other experimental work,acylating agents were added in therate of 0.5 g/minute.

Acetylation of Secondary Groups

The relative reactivities of second­ary groups are shown in Figures 2 to5. The general features of the acetyla­tion of total secondary groups were

100

20

"g 60.~

>-E 40

"

100

80

M ~ ~ W 100Conc. of acetic anhydride (mM)

1-15 1:8 1:5 1:4 1:3Anhydride to protein (w/wl

Cone. of anhydride (mMJ

1.8 1 5 1:4

Anhydnde to protein {w/wl

1 15

80

100

1.3

100806040

Cone. of anhydride (mMI

1.8 1:5 1:4Anhydride to protein (w/wl

20

us

"~ 60

.~>-E 40

"

80

Figure I.-The reactivity of aminogroups of fish myofibrillar proteinduring acetylation with aceticanhydride (protein content,30.2-31.6 mg/g).

Figure 2.-0verall reactivity of thetotal secondary groups of fishmyofibrillar protein with aceticanhydride (protein content,30.2-316 mg/g).

Figure 3.-The reactivity of tyrosylgroup of fish myofibrillar proteinwith acetic anhydride (protein con­tent, 30.2-31.6 mg/g).

16 Marine Fisheries Review

similar to the acetylation of the aminogroup, except that the reaction ratewas greatly reduced at lower pH's(Fig. 2). For example, at 40 mManhydride, about 35, 25, and 20 per­cent of total secondary groups werereacted at pH 9.5, 7.5, and 6.5,respectively, while at least 65 to 90percent of the amino groups wereacylated at the same anhydride con­centration.

The rates of acetylation of second­ary groups at pH 6.5 and 7.5 ap­peared to be relatively linear;however, at pH 9.5, concentration ofacetic anhydride between 40 to 70rnM increased the rate considerably.When Figures 1 and 2 are considered,it can be seen that total acylation atsecondary residues of protein mightbe kept relatively low by controlleduse of anhydride and somewhatminimized by acetylating at pH 9.5.On the other hand, relatively highlevels of acetylation of the secondarygroups can only be accomplishedafter the acetylation of the aminogroup is almost complete.

Acetylation of Tyrosyl Group

As shown in Figure 3, the phenolicgroup of tyrosine can be acetylated toa level of more than 90 percent by us­ing a high concentration of anhydrideand extended reaction time. The reac­tivity of the tyrosyl group wasrelatively low at lower levels of

100

80

20

40 60 80 100Cone. of acetic anhydride (mM)

115 L8 1:5 1:4 1:3Anhydride to protein (w/w)

Figure 4. -The reactivity of sulf­hydryl group of fish myofibrillarprotein with acetic anhydride (pro­tein content, 30.2-31.6 mg/g).

March 1981, 43(3)

reagent. For example, at 40 mManhydride concentration, approx­imately 30 percent of tyrosyl groupswere reacted; and at above 50 mM ofanhydride at pH 6.5, the reaction ratedecreased considerably. Again, it isnoticeable that the rate of the reactiontended to accelerate dramatically atpH 9.5 and 7.5 with 40 to 60 mManhydride, at which point the acetyla­tion of amino groups was nearly com­plete. This tendency was revealedmore clearly on tyrosyl groups than inthe overall reaction of total secondarygroups because the acylation oftyrosyl groups is quantitatively muchgreater than acylation of sulfhydryland hydroxyl groups, particularly athigher concentration of anhydride.The pH dependence of the reactionwas not as great between pH 7.5 and9.5 as between pH 7.5 and 6.5, andthis effect was more evident at higherconcentrations of anhydride than atlower levels.

Acetylation of Sulfhydryl Group

Unlike tyrosyl or hydroxyl residues,the sulfhydryl group of fish proteinshowed a high reactivity with aceticanhydride even at lower levels ofanhydride concentration, yieldingover 50 percent acetylation with 40rnM anhydride (ratio to protein, 1:8)at both pH 9.5 and 7.5 (Fig. 4). Thepattern of the reaction was also dif­ferent from those of the other residues

100

80

~ 60o.~

>-E 40

"20

O"'------",---------L_---I._---I._---'-,-----~

20 40 60 80 100Conc. of acetic anhydride (mMl

1:15 1.8 1:5 1:4 1:3Anhydride to protein (w/wl

Figure 5.-The reactivity of hydrox­yl group of fish myofibrillar proteinwith acetic anhydride (protein con­tent, 30.2-31.6 mg/g).

in that the overall reaction proceededsimilarly to the amino group reaction.The reaction rate of pH 6.5 wassignificantly less than at pH 7.5 and9.5 with anhydride levels greater than30 mM (ratio to protein, I: 10). Arelatively high acylation reactivity ofsulfhydryl in papain compared withmore simple compounds has beenreported by Finkle and Smith (1958),Wallenfels and Eisele (1968), andChaiken and Smith (l969a). Chaikenand Smith (1969b) suggested that anexplanation for the high rate of reac­tion of the sulfhydryl group withsome acylating agents may be intra­protein interactions involving thesulfhydryl, imidazolium, and car­boxylate groups of the protein whichmay cause increased nucleophility ofthe sulfur anion.

Acylation of Hydroxyl Group

It has been generally known thatthe hydroxyl groups of serine andthreonine show the lowest reactivitywith acetic anhydride compared withother secondary side groups. Thereaction rates at pH 6.5 and 7.5decrease at about 30 mM anhydrideand lower concentrations and in­crease at high levels of anhydrideconcentration. The reaction rate atpH 9.5 is relatively linear up to 100mM anhydride. No more than 28 per­cent was modified with 40 mManhydride at pH 9.5 (Fig. 5).

Inhibitory Effectof Sodium Sulfite

It is important to know if the reac­tion of acylating agents with thesecondary functional groups on pro­tein can be modified by use ofchemicals that might be acceptablefood additives. Sulfite, a widely usedadditive, has been shown byPfleiderer et al. (1968) to protect all ofthe sulfhydryl groups in lactatedehydrogenase during the acetylationwith acetylimidazole.

To evaluate the inhibitory effect ofsodium sulfite on acylation of second­ary groups, fish myofibrillar proteinwas acylated with acetic and succinicanhydride in the presence of up to 20mM sulfite. As the results of Figures 1

17

Table 5.-Thalnfluance of pH on the retardation of the acetylation of fish myofibrlller protein in the presence ofsulfite.

Percent acetylation on:

Sodium Acetic Totalsulfite anhydride Amino secondary Tyrosyl Sulfhydryl Hydroxyl

pH (mM) (mM) group groups group group group

6.5 15 60 33.2 9.2 0 36.8 11.77.5 15 60 75.0 12.9 5.2 37.8 14.09.5 15 60 79.6 19.2 7.0 41.5 22.6

Table 4.-Elfect of sulfite on succlnylatlon of functional groups In fishmyoflbrlllar protein at pH 7.5.

Percent succinylation on:

Sodium Acetic Total Sulf·sultite anhydride Amino secondary Tyrosyl hydryl Hydroxyl(mM) (mM) group groups group group group

0 95 80.8 6.6 0 36.6 6.75 86 70.9 5.6 0 28.0 6.0

10 86 66.3 5.4 0 15.9 6.215 86 51.2 4.1 0 15.9 4.8

Table 3.-Elfect of sulfite on acetylation of functional groups In fishmyoflbrlllar protein at pH 7.5.

Percent acetylation on:

Sodium Acetic Total Suit·sulfite anhydride Amino secondary Tyrosyl hydryl Hydroxyl(mM) (mM) group groups group group group

0 100 94.8 78.2 95.1 95.1 69.75 97 88.4 28.7 26.5 46.3 28.0

10 106 83.8 26.2 13.2 40.2 30.715 107 78.3 22.2 0 40.2 30.320 107 75.0 20.8 0 39.0 28.3

15 rnM sulfite at pH 9.5 (Table 5);however, acetylation was reduced 24percent on secondary groups that in­clude 60 percent reduction on tyrosyland 13 percent on sulfhydryl, but littleor no change in hydroxyl.

Discussion

The results of acetylation withacetic anhydride show that the aminogroups of fish myofibrillar proteinswere readily acetylated to a level of80-90 percent under relatively mildconditions. However, the overall reac­tivity of secondary groups, particular­ly hydroxyl, was much less than thatof the amino group, a function of pHand reagent concentration. The effectof pH on the reaction rate of protein

were only minor changes forsulfhydryl and hydroxyl.

From these results it is concludedthat sulfite can be used effectively toretard acylation of secondary groups;however, it may be less useful for suc­cinylation reactions since only thesulfhydryl reaction is significantlyretarded with the acylation agent. Forexample, acetylation with an an­hydride to protein ratio of 1: 10 at pH9.5 results in 80 percent acetylation onamino and 25 percent on total second­ary groups that include 18 percenton tyrosyl, 46 percent on sulfhydryl,and 22 percent on hydroxyl (Fig. 1).The same level of acetylation on ami­no group was obtained at an an­hydride to protein ratio of 1:5 with

and 2 and Table 3 indicate, sodiumsulfite was effective in retarding thereaction with sulfhydryl, tyrosyl, andthe amino group. In contrast to theresult of Pfleiderer et al. (1968),however, the sulfite was more effec­tive in retarding the reaction oftyrosyl than the sulfhydryl group.There was decreased acylation of theamino group and protection of thehydroxyl and sulfhydryl groups. Ingeneral, the protection effect was in­creased with higher levels of sodiumsulfite (Table 3). However, the effectdiffered depending on the kinds ofsecondary group. For example, acyla­tion of tyrosyl groups was completelyinhibited at a level of 15 rnM ofsodium sulfite while acylation ofsulfhydryl and hydroxyl was reducedto approximately 50 percent at 5 rnMof the sulfite and higher levels ofsulfite did not retard the reaction fur­ther. The retardation effect wasgreater for the secondary groups thanfor the amino group. At a level of 15mM sulfite, acetylation was reduced20 percent on the amino group, 70percent for total secondary groups, 60percent on the sulfhydryl, 55 percenton the hydroxyl, and 100 percent onthe tyrosyl groups.

Sulfite, as shown in Table 4, wasalso effective in reducing the suc­cinylation of protein functionalgroups. A significant retardation ofsuccinylation of the sulfhydryl groupoccured in the presence of sulfite,while the reaction of the hydroxylgroup was not greatly affected. Suc­cinylation of the tyrosyl group wasnot detected as other investigatorshave reported earlier (Riordan andVallee, 1964; Gounaris and Perlmann,1967). Although the extent of suc­cinylation was lower than acetylation,the protective effect was revealedalmost the same as in the case ofacetylation. Fifteen rnM sulfite reduc­ed succinylation 37 percent on amino,60 percent on sulfhydryl, and 28 per­cent on hydroxyl groups.

The pH dependence of the retarda­tion effect of sodium sulfite is shownin Table 5. A major decrease in theacetylation of amino and tyrosyl wasshown below pH 7.5; however, there

18 Marine Fisheries Review

residues tended to reflect, as generallyrecognized, that the acetylation reac­tion was more favorable at pH 9.5than at pH 7.5 or 6.5. The pH effecton the reaction rate was greater on thesecondary groups than on the aminogroup at higher levels of anhydride,whereas at lower levels of anhydrideconcentration, the differences werevery small.

Tyrosyl showed a large increase inacetylation rate at higher levels ofanhydride. Also, a large decrease inthe acetylation of tyrosyl occurred inthe presence of sulfite. This large in­crease in acetylation rate might be ex­plained by exposure of buried tyrosylgroups as a result of acetylation­induced conformational changes inthe protein (Habeeb, 1966). Thedecrease in the acetylation of tyrosylin the presence of sulfite might be dueto a sulfite-induced conformationchange in protein as was suggested byPfleiderer et al. (1968).

Conclusions

Protein amino groups were readilyacylated under mild conditions at pH9.5 to 6.5 with 30 to 50 roM anhydride(ratio of anhydride to protein, 1: 10 to1:5, weight:weight) added at the rateof 0.5 g/minute resulting in 80-90percent acetylation. Under the sameconditions, the reactivities of second­ary groups were much less; only 20-30percent as total secondary group,20-35 percent of tyrosyl, 40 percent ofsulfuydryl, and 20 percent of hydrox­yl groups were acetylated. Sodiumsulfite was effective in retarding theacylation of these secondary groups.Fifteen roM sulfite reduced acylationby 20 percent on amino, 60 percent onsulfuydryl, 100 percent on tyrosyl,

March /98/, 43(3)

and 55 percent on hydroxyl groups;and 15 roM sulfite reduced succinyla­lion by 37 percent on amino, 60 per­cent on sulfuydryl, and 28 percent onthe hydroxyl group.

The relative acylation reactivities ofprotein groups might be considered interms of the modification of proteinsfor food use. In much of the reportedwork on food-protein modification,about 80 percent acylation of theamino group is indicated. If 80 per­cent acylation of the amino group isused as an example and if minimalamounts of secondary products aredesired, it is recommended that thereaction conditions be pH 9.5 with ananhydride to protein ratio of 1: 10.Under these conditions, acylationwould result on 20-30 percent of thetotal secondary groups that include20-30 percent tyrosyl, 40 percentsulfuydryl, and 20 percent hydroxylgroups. It is further recommendedthat when the need for the leastsecondary product is indicated, thenthe acylation should be performed inthe presence of sulfite.

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