ABSTRACT. Kinetic parameters for heat-induced denaturation of lactoferrin under different conditions were determined over a temperature range 72-.
Kinetic Parameters for Denaturation Bovine Milk Lactoferrin L. SANCHEZ,
J.M. PEIR6,
H. CASTILLO,
ABSTRACT Kinetic parameters for heat-induced denaturation of lactoferrin under different conditions were determined over a temperature range 7285°C. Denaturation of lactoferrin could be described by first-order reaction kinetics. Lactoferrin is denatured more rapidly in its apo form than in the iron-saturated form. Both apolactoferrin and iron-saturated lactoferrin are more heat-sensitive when treated in milk than in phosphate buffer. Values of change in enthalpy of activation of lactoferrin denaturation are high which indicates that a large number of bonds are broken. The association of lactoferrin with 8-lactoglobulin does not significantly influence the change in enthalpy of activation of lactoferrin denaturation. Key Words: DB, milk, kinetics, denaturation, lactoferrin
INTRODUCTION
LACTOFERRINis a glycoproteincharacterized by its reversible iron-bindingcapacity. Crystallographicanalysishas revealedthat lactoferrinconsistsof a single polypeptidechain structuredin two globularlobes, correspondingto the N- and C- terminal halvesof the molecule(Andersonet al., 1987). Eachlobecontainsoneiron-bindingsite in an interdomaincleft (Bakeret al., 1987).Dueto the strongly anionic characterof this site cationswith high positive chargeare tightly bound, thus Fe3+ binds more strongly to lactoferrin than Fe*+ (Brock,
1985). The atom of iron is bound to lactoferrin through its interactionwith four ligands:two tyrosines,one asparticacid andonehistidine(Andersonet al., 1989).A bicarbonateanion binds simultaneouslywith an atom of iron and moleculesof water also appearto be involved in the binding (Andersonet al., 1989). The binding of iron to lactoferrincausesa conformational change,the moleculebecomingmorecompact.The iron enters into the open interdomaincleft in eachlobe and then the domainscloseover the iron atom (Andersonet al., 1989).This changein lactoferrinstructureexplainswhy iron-saturated lactoferrin is more resistantto denaturationand proteolysisthan the apo form, (especiallythe C-lobewhich is more compact than the N-lobe, Anderson,1990). Lactoferrinhasbeenfoundin manyexternalsecretions,such asmilk, bronchialsecretions,andsaliva(AisenandListowsky, 1980),andin the secondarygranulesof neutrophils(Miyauchi and Watanabe,1987). Due to its high isoelectricpoint, lactoferrin can associatewith other macromolecules,especially thoseof acidiccharacter(Hekman,1971).In milk, it associates with proteinssuch as IgA secretorycomponent(Watanabeet al., 1984),caseinand albumin(Hekman,1971)lysozymeand someglycopeptides(Jorieuxet al., 1985)and P-lactoglobulin (Lampreaveet al., 1990). Lactoferrinhasbeenisolatedfrom milk of manyspeciesand is in the highestconcentrationin colostrum(MassonandHeremans, 1971). In bovine milk the concentrationof lactoferrin decreasesto a very low level in the first days of lactation (Sanchezet al., 1988).However,in humanmilk the concenThe authors are with Tecnologla y Bioquimica de /OS Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet, 177, 50013 Zaragoza. Spain. Address inquiries to Dr. M. Calvo
M.D. PliREZ,
of
J.M. ENA, and M. CALVO
tration of lactoferrin is high throughoutthe lactationperiod (Houghtonet al., 1985)which has led to suggestionsthat this protein could have importantfunctionsin the newborn.Lactoferrin exhibits bacteriostaticactivity, depriving bacteriaof iron, and could be involved in the protectionof the newborn againstinfections,althoughin vivo evidenceis lacking (Moreau et al., 1983). It has also beensuggestedthat lactoferrin could participatein some functions of the immune system, such as granulopoiesis(Broxmeyeret al., 1984). The high bioavailability of iron from humanmilk suggestsa role for lactoferriniron absorptionin the intestine(Cox et al., 1979; Franssonet al., 1983a,b). This could be mediatedthrough interactionof lactoferrinwith a receptorin the intestine,the existenceof which has beensuggestedin somespecies(Davidson and Lijnnderdal,1988, 1989; Hu et al., 1990). Lactoferrin may also affect control of cell growth, althoughit has not yet beenelucidatedwhetherthis is due to the iron bound to lactoferrin or to some other mechanism(Nichols et al., 1987, 1989). Humanbreast-feedingis commonlysubstitutedby formula milks which are manufacturedmainly from cows’milk. This milk is subjectedto severalmodificationsin its composition to makeit as similar as possibleto humanmilk. It is therefore importantto fully characterizethe componentsof humanand bovine milk.
Moreover,
it is also necessary to know what
effect heattreatmenthason the integrity of milk components. Although many studieshavebeenconductedon heat-induced denaturationof milk proteins(Dannenberg and Kessler,1988; Dalgleish, 1990; Paulssonand Dejmek, 1990),there are discrepanciesin resultsdueprobablyto the useof differentmedia to treatproteinsanddifferentmethodsto measuredenaturation. The objective of our work was to determinethe rate of thermal destructionof lactoferrin in different conditions, in order to facilitate the designof heat treatmentsthat preserve its integrity and thus its possiblebiologicalactivity. MATERIALS
& METHODS
Materials Bovine lactoferrin was provided by Fina Research (Seneffe, Belgium) and bovine 8-lactoglobulin (a mixture of A and B variants) was supplied by Sigma (Poole, Dorset, England). Cows’ milk was sunolied by Tauste-Gaiadera (Zaragoza, Spa&). It’was skimmed by cen~rifugation at 2000 x IE for 15 min at 4°C. In order to eliminate endorrenous Lctoferrin, milk was immuno-adsorbed on a column of anti-la&ferrin antibodies insolubilized on CNBr-activated Sepharose according to Fuchs and Sela (1986). Modification of lactoferrin iron-saturation Lactoferrin was iron-saturated with 10 mM ferric nitriloacetate (FeNTA) and solutions at 0.2 mg/mL were prepared in O.OlM phosphate 0.15M NaCl buffer, pH 7.4, or in immuno-adsorbed cows’ milk. Lactoferrin was iron-depleted according to the method of Mazurier and Spik (1980). A lactoferrin solution of 1 mg/mL was dialyzed overnight vs. 0.2M acetic acid/sodium acetate, 40 mM EDTA, 0.2 M sodium phosphate, pH 4, then dialyzed vs. phosphate buffer and filtered through a 0.22~urn filter. This solution was then concentrated using Amicon Diaflo ultrafiltration membrane cones CF25. Glass capillaries and all the material in contact with the apolactoferrin soVolume
57, No, 4, 1992-JOURNAL
OF FOOD SCIENCE-873
DENATURATION OF LACTOFERRIN... proteins after thermal treatment denatured samples were cooled at WC/min to 30°C and rescanned. Thermoresistometer. The change in pH of bovine skimmed milk and 0.01 M phosphate, 0.15M NaCl buffer, pH 7.4, over a SO-130°C temperature range was studied in a thermoresistometer (TR-SC) designed in our laboratory by Cond6n et al. (1989). The pH of both solutions was measured 10 rnin after temperature stabilization.
7.67.4
-
7.2
-
7.0
-
6.8
-
6.6
6.2
-
6.0
-
6.4
Calculation of D and Z values
werecalculated, by D values(timerequiredfor 90% denaturation) regression analysis,asthereciprocalof theslopecorresponding to the linesobtainedby plottingthe logarithmof the concentration at each
5.85.6
-
,.*
I 40
60
80
&I
40
tl0
TEMPERATURE (*C) Fig. 1 -Effect of temperature on pH of bovine milk (0) end phosphate buffer (A).
lution were previously soaked in O.lN HCI overnight, to remove iron contamination, and exhaustively washed with deionized water. Immunochemical
techniques
Antisera preparation. Antiserum to anti-lactoferrin was developed in rabbits which were immunized by several subcutaneous injections in the back with 2 mg pure lactoferrin in 0.5 mL of O.OlM phosphate 0.15M NaCl buffer,pH 7.4, homogenized with 0.5 mL of complete Freund’s adiuvant. After 4 wk the rabbits were immunized again with the same amount of protein. Ten days later the animals \;ere bled from the ear vein and the antibodies tested by immunodiffusion. When the serum gave a positive reaction against lactoferrin the rabbits were bled by cardiac puncture. Specificity of the antisera was checked by immunoelectrophoresis. Measurement of lactoferrin concentration. Lactoferrin concentration was determined by radial irnmunodiffusion according to Mancini et al. (1965). Agarose (1% in 0.025M barbital buffer 0.3M NaCl, pH 8.2) was prepared containing 0.5% specific antiserum and spread onto 9 x 12 cm glass plates precoated with 0.8% agarose in distilled water. The solidified gel was 2 mm thick and twenty wells 2 mm in diameter were cut out. Samples (5 pL) and appropriate standards of pure lactoferrin were added to the wells and the plates were incubated in humidified chambers at room temperature (-22°C) for 72 hr. After immunodiffusion, the plates were washed in phosphate buffer for 24 hr with frequent changes. Finally, the plates were washed once with distilled water and allowed to ah-dry. The gel was stained for 20 min with Coomassie Blue (250 mz/L) in methanol/distilled water/ acetic acid (4.5:49:6) and then destainid in methanol/acetic acid/ glyc; erol/dbtilled water (25:8:2:65). Standard curves were made by plotting the square of the diameter of the precipitating rings vs. concentration. Heat treatment Capillary method. Lactoferrin solutions (2Op,L) were introduced into glass capillary tubes (1.5-1.6 mm outer diameter, 1.1-1.2 mm inner diameter) which were sealed with a microflame and checked by immersion in water. The capillaries were immersed in a temperaturecontrolled water bath (*O.l”C) at four different temperatures: 72,77, 81 and 85°C. Heated samples, in duplicate, were removedfrom the bath at different intervals and immediately cooled by immersion in an ice water bath. Undenatured lactoferrin was quantified by radial immunodiffusion as described above. of apclactofenin, 30% Differential scanning calorimetry. Solutions and 100% iron-saturated lactoferrin and g-lactoglobulin were prepared in O.lM phosphate 0.15 M NaCl buffer, pH 7.4, at a protein concentration of 80 mg/mL. Samples of 15 FL (1.2 mg of protein) of the different solutions were pressure-sealed in DuPont aluminum pans. To check hermetic seals, pans were subjected to a vacuum and weighed with an electronic microbalance. The reference samples consisted of identical pans filled with a similar weights of phosphate buffer solution. DSC thermograms were recorded on a DuPont model DSC 10 thermal analyzer programmed for a heating rate of 10”C/min in the temperature range from 35 to 110°C. To estimate renaturation of the
874-JOURNAL
OF FOOD SCIENCE-Volume
57, No. 4, 1992
treatment as a function of time. The effect of temperature on D value was also studied and the Z value (degrees necessary to reduce D value cycle)wascalculated by regression analysis.Z was in one logarithmic obtained from the slope of the line obtained when plotting D values vs the corresponding temperatures.
RESULTS & DISCUSSION Change of milk and buffer pH with temperature
It is well known that variation in temperature produces changes in the pH of solutions, dependingon their composition(Walstra and Jenness,1984). We found (Fig. 1) that bovine milk showsa greaterchangein pH with an increaseof temperature than phosphatebuffer, especiallyabove90°C. However, over the temperaturerange in which the-thermoresistance of lactoferrin was studied (72-85”C) the changeof pH was slight. In the temperaturerangebetween20°C and 85”C, milk pH varied from 6.6 to about 6.2 and that of the buffer from 7.4 to about 7.2. Denaturation
of bovine lactoferrin
As a first step, preliminary experimentswere designedto determinean appropriatetemperaturerangeto study lactoferrin denaturationby the capillary method. We observedthat ironsaturatedbovine lactoferrin in phosphatebuffer denaturedvery slowly at 65°C. Treatmentat that temperaturefor 45 min caused only 30% denaturationof lactoferrin and thus a temperature range of 72-8S’C was chosen. The degreeof lactoferrin denaturationincreasedwith temperatureand time of treatment(Fig 2) we also observedthat the time of denaturationfor a given temperaturewas shorter for apolactoferrinthan for iron-saturatedlactoferrin and that both were more heat-labilewhen they were treated in milk than in phosphatebuffer. The graphsshow resultsof a single experiment,althoughall assayswere in duplicateor triplicate and meanvalueswere used to calculateall the kinetic parameters. D and Z valueswere calculatedas describedin the methods section(Table 1). D value changedasa function of temperature (Fig. 3). The D value decreasedwith temperatureand was higherfor iron-saturatedlactoferrinthan for apolactoferrin,and higher when both were in phosphatebuffer than in milk. The difference in thermoresistanceof both forms of lactoferrin, with andwithout iron, was probablydue to the conformational change that occurs when lactoferrin binds iron. Lactoferrin domainsclose over the interdomaincleft where the iron-binding site is located,making the moleculemore compact(Baker et al., 1987). Transferrin, structurally similar to lactoferrin, has also been reported more resistant to denaturationwhen iron-saturated(Baker et al., 1987). The thermoresistanceof lactoferrin, either iron-saturatedor iron-free, is greaterwhen treated in phosphatebuffer than in milk. This might be attributedto changesin the calcium phosphate bound to caseinswhich changesto a more amorphous statewith increasingtemperature.Thus, interactionsof lactoferrin with caseinswould be enhancedand, as a consequence, lactoferrin heat sensitivity increased(Visser et al., 1986). In addition,
the presence of or-casein decreases the temperature
3.0
72X!
1
,3 , , , , , , , , V.”
.
0
500
lcoo
1500
2Cm
Holding
2500
3Oc0
3500
0
4ooo
500
1500
loo0
time (set)
20&l
Holding
2500
3000
3500
3.0 2.5
4cOO
time (set)
85’C
2.5 2.0 L "
1.5
i! 1.0 0.5
0.5
0.0 0
1000
1500
Holding
time (set)
500
2000
0.0
2500
500
0
Ical
1500
Holding
time (set)
Fig. 2-Effect of heat treatment on the denaturation of iron-saturated lactoferrin (04 and apolactoferrin buffer (AA) at different temperatures. c, is undenatured protein concentration at each holding time.
Table I-D and Z values for iron-saturated lactoferrin end apolactoferrin at different temoeratures in ohosohate buffer and in milk
072 D77 DE4 D8S 2
20 10 3.5 1.7
x x x x 13
103 103 103 103
Apolactoferrin 6.5 4.7 1.9 1.3
5.5
Milk
Phosphatebuffer Saturated
6.0
x x x x 17
lo3 103 103 103
Saturated 4.5 3.1 1.2 6.5
x x x x 15
10" 103 103 102
Apolactoferrin 1.3 1 4.3 2.5
x x x x 17
2c00
2500
(oA) in milk (00) or phosphate
1
103 103 102 lo2
of denaturationof P-lactoglobulin,or-lactalbumin and albumin when measuredby differentialscanningcalorimetry(Paulsson andDejmek, 1990).Interactionof lactoferrinwith otherwhey proteinsmay also modify the thermoresistance of lactoferrin. The greaterdecreaseof milk pH, in comparisonwith that of phosphatebuffer, could also contributeto the low thermoresistanceof lactoferrinwhen treatedin milk. The denaturationof milk proteinsin a solutionwith an ionic compositionsimilar to that of milk has been studied(Riiegg et al., 1977).Suchsolutionhas the deficiencyof lacking caseinsandwhey proteinsandthusthe interactionsbetweenthem which could influencetheir heat sensitivity.Therefore,those resultscannotbe extrapolatedto effectsin whole milk when it is heat-treated. Somestudieshavebeencarriedout on the thermoresistance of lactoferrinin humanmilk, but with contradictoryresults. Ford et al. (1977)found that, at 7o”C, treatmentfor 15 set caused95% of lactoferrinto be denatured,while Goldblumet al. (1984) reportedthat 100% of lactoferrinremainedintact when treatedat the sametemperatureand holding time. Our results agreedwith theirs. The different resultsobtainedby Ford et al. (1977)could be attributedto the techniqueof electro-immunodiffusionusedto measurelactoferrinconcentration after treatment.That techniquecould lead to considerablein-
1.5 I 1.0:
. 65
I 70
.
I 75
.
I 80
TEMPERATURE
’
I 85
.
I 90
.
I 95
(‘C)
Fig J-Effect of temperature on Dt values (time for 90% denaturation) of the denaturation of saturated lactoferrin(*A) and apolactoferrin (oa) in phosphate buffer (A/L) or in milk (00).
accuraciesbecause,at the pH employed,lactoferrin hardly migratesdue to its high isoelectricpoint. Kinetic analysis Order of reaction. The concentrationof undenatured lactoferrin at each time of treatmentwas subjectedto reaction kinetics analysis.The reactionof denaturationof a protein behavesin an analogousway to a general-rate reactionof order n accordingto the equation
Volume 57, No. 4, 1992-JOURNAL OF FOOD SCIENCE-875
DENATURATION OF LACTOFERRIN... a.
b.
0
500
lcoo
1500
2ooo
Holding
2500
3mo
3500
4ccKl
time (set)
0
500
lcoo
1500
2ooo
Holding
2500
3coo
35ccl
4ooo
time (see)
3.0 2.5 _
2.0
u o
1.5
"
: 1.0 0.5 0.0 0
500
loo0
1500
2ooO
Holding
2500
time
3OW
3500
4ooO
(set)
0
500
loo0
1500
2000
Holding
time
2500
3OC0 3500
4000
(set)
Fig. I-Denaturation of iron-saturated lactoferrin /@Am+) and apolactoferrn @An 0) in phosphate buffer (a) or in milk (b) as a firstorder reaction at 72°C (o), 7PC (A), Sl’@) and 85°C I+). c, is the initial concentration of protein and c, the undenaturated protein concentration at each holding time.
Table 2-Results of the application of kinetic parameters to the denaturation of iron-saturated lactoferrin and apolactoferrin in phosphate buffer and in milk at different temperatures K(x104)
Sat.
r
b Apo.
Sat.
Apo.
Sat.
Apo.
Phosphatebuffer 72°C 77T 81'C 85°C 72°C 77°C 81°C 85%
1.2 t-i 1io
4.6 5.2 19.1 22.5
0.009 0.023 0.080 0.049
0.256 0.113 0.170 0.138
0.986 0.971 0.949 0.979
0.885 0.952 0.933 0.942
4.7 8.1 18.4 56.9
12.2 20.4 54.4 91.2
0.055 0.125 0.055 0.048
0.030 0.055 0.030 0.161
0.980 0.980 0.983 0.985
0.988 0.980 0.988 0.926
-dc/dt = kc”
(1)
where -dc/dt representsthe rate of protein denaturation,k the rate constant,c the protein concentrationat eachtime, and n the order of reaction. Consideringthe order of reaction as 1, the equation (1) is then -dc/dt = kc, -de/c = kt and integrating it In cJct = kt is obtained, where c, is the initial concentrationand c, the concentrationat each holding time. When the experimentalpoints are plotted accordingto this last equationstraight lines are obtainedby linear regressionfrom which the rate constantk from the slope, the coefficient of correlationCzand the value of the ordinate interceptb (time t = 0) are calculated.The graphicalrepresentationof the denaturation of lactoferrin in different conditions as a first-order reactionis shown (Fig. 4) and the values of k, b and ti (Table 2). The straight lines show a high coefficient of correlation. 876-JOURNAL
OF FOOD SCIENCE-Volume
57, No. 4, 1992
They intersectthe ordinatenear0 which indicatesthat the value of n = 1 is suitable, at the temperaturesstudied, to describe mathematicallythe reaction of lactoferrin denaturation.However, for apolactoferrintreatedin phosphatebuffer the correspondinglines intersectthe ordinatefurther from 0. This might indicate some aggregationduring denaturation,as has been describedfor p-lactoglobulin (Harwalkar, 1980a,b). Kinetic parameters. The temperatureof treatmentand the rate constantin a denaturationprocessare relatedaccordingto the Arrhenius equation: k = Ke-E.PT,
where k is the rate constant, K a constant,EA the apparent activation energy, R the universal gas constant, and T the absolutetemperature.When the rate constantwas plotted vs. the reciprocalof the absolutetemperaturea linear relationship was observedin the temperaturerange studiedwhich allowed determinationof the activation energyvalues (Table 3). This relationshiphas also been found to be linear for the denaturation of other milk proteins, althoughin someof them a break in linearity with a changein the slope has been reportedfor temperaturesabove80-90°C (Dannenbergand Kessler, 1988). High valueswere found for the activationenergy,indicating that a large number of bonds are broken during lactoferrin denaturation,since the energy of each of them is low. However, we found that the activation energy for iron-saturated lactoferrinwas higher than for apolactoferrin.This may be due to the greater stability of iron-saturatedlactoferrin, a larger amountof energybeing necessaryin order to activatethe molecule and start denaturation.
-6 x
-7-
M e ,-
E -8 -
-10 2.76
I 2.60
I 2.82
I 2.84
1 2.86
I 2.88
1 2.90
I 2.92
-7-8 -
-10 2.78
.
( 2.80
.
, 2.82
l/T (x 10 3 )
g
10 3
(X
., 2.88
., 2.90
2.92
j
-7-
9
-8 -
-8 -
-9 -
2.78
., 2.86
-6 -
-7-
-10
., 2.84
’ l/T
-6 * +
.,
-9 I 2.80
I 2.82
I 2.84
I 2.86
1 2.88
I 2.90
I 2.92
-IO2.78
.
I 2.80 ,
. 2.82 ,
l/T (x lo3 ) Fig. 5-Effect of temperature on the rate constant phosphate buffer (~a or in milk (00). l/T represents
, 2.84 ,
2.m'2
l/T (x 10 3 ) (k) of denaturation of iron-saturated lactoferrin the reciprocal of the absolute temperature.
The value of the activationenergyalloweddeterminationof different thermodynamicparameterssuch as the enthalpy,the entropy and the free energyof activation (AH#, AS# and AG#, respectively)accordingto the following expressions: AH# = E,-RT
apolactoferrin
(oa)
in
Table 3-Change in enthalpy of activation. AH#, free energy of activation, AG#, and entropy of activation, AS#, at different temperatures for denaturation of iron-saturated lactoferrin and apolactoferrin in phosphate buffer and in milk Phosphate
buffer
SATURATED EA = 202.79 kJ.mol-1
AS# = R (In A - In K$ h, - In T)
AG# = AH# - T AS# whereR is the universalgas constant,In A the ordinateintersectionwhen regressionanalysisis appliedto the plot obtained in the calculationof activation energy, KB is the Boltzmann constant,h, the Planck constant,and T the absolutetemperature. The samevaluesfor thesekinetic parameterswere obtained at the different temperaturesstudied,(Table3). The high valuesof the enthalpyof activationand the positivevaluesof the entropy of activation indicate that, during denaturation,lactoferrin experiencesa largechangein conformation.However, our valueswere lower than thoseobtainedfor other milk proteins such as B-lactoglobulinA and B and cx-lactalbumin,for which the enthalpyof activation is 260-280 kJ/mol and the entropy of activation 0.4-0.5 kJ mol-‘K-r in this rangeof temperature(Dannenbergand Kessler, 1988). The values obtainedfor thoseparameterswere still lower for apolactoferrin thanfor the iron-saturated form, althoughthey arestill constant at all the temperatures studied.This is probablydueto the less stableconformationof lactoferrinwithout boundiron. Differential scanning calorimetry. Resultsof differential scanningcalorimetry(DSC) of lactoferrinat different degrees of iron saturation,B-lactoglobulinand a mixture of both proteins are shownin Fig. 6 and Table4. A large differencewas observedbetweenthe endotherms(peaksof heat absorption) of apolactoferrinandiron-saturated lactoferrinobtainedby DSC. When 100% iron-saturatedlactoferrin was denaturedan en-
(@A) and
72°C 77°C 81°C 85°C
APOLACTOFERRIN EA = 138.92 kJ.mol-1
AH#
AG#
AS#
AH#
AG#
AS#
199.92 199.88 199.85 199.81
108.15 106.78 105.69 104.58
0.286 0.266 0.266 0.266
134.05 134.01 133.98 133.94
104.38 103.91 103.89 103.51
0.086 0.086 0.085 0.085
Milk SATURATED EA = 183.97 kJ.mol-' 72°C 77°C 81°C 85°C
181.10 181.06 181.03 180.99
104.51 103.36 102.44 101.51
APOLACTOFERRIN EA = 157.51 kJ.mol-1 0.222 0.222 0.222 0.222
154.64 154.60 154.57 154.53
101.51 100.70 100.05 99.40
0.154 0.154 0.154 0.154
dothermwith two peakswas found,with temperatures of maximum heat absorption(also referred to as temperatureof denaturation)at 74°C and 86S”C. The appearance of the two peaksmight be attributedto different heatsensitivity between the 2 lobesof lactoferrin, sincethe C-lobemay be more compact than the N-lobe (Andersonet al., 1989).This difference could also be due to the formation of monoferric speciesif iron bound to lactoferrin is sequestered by phosphatewhen temperatureincreases(Riiegg et al., 1977). The first hypothesis seemsmore likely and is supportedby resultsreportedby Evansand Williams (1978). They reporteda higher temperature of denaturationfor the C-lobe of transferrinthan for the N-lobe when measuredby DSC. Volume
57, No. 4, 1992-JOURNAL
OF FOOD
SCIENCE-877
DENATURATION OF LACTOFERRIN...
I 20
40
60
80
TEMPERATURE
100
120
(‘C)
bound iron have the same thermosensitivityand denaturesimultaneously.Renaturationwas not observedfor any of the samplesanalyzedby DSC. The integration of the endothermsgives the value of the enthalpyof denaturation,(Table 4). This parameterreflects a net absorptionof energy. That is, the result of the energy necessaryto break the hydrogenbondswithin the protein and those that are formed with water; the energy liberatedwhen new bonds are formed between the protein and water, and betweenthe moleculesof water aroundthe apolargroups; and the energy necessaryto break van der Waals forces between apolargroups,which is almostnegligible(Pa&on andDejmek, 1990). In DSC analysis of lactoferrin we observedthat the enthalpyof denaturationincreasedwith iron saturationreflecting a correspondingincreasein stability. It is well known that lactoferrin interactswith some milk proteinssuch as @lactoglobulin(Lampreaveet al., 1990) and it would be possiblethat such interactionscould have an influence on lactoferrin denaturation.However, we found that the enthalpyof denaturationfor the mixture of S-lactoglobulin and 100% iron-saturatedlactoferrin correspondsto the sum of the enthalpiesof denaturationfor eachindividual protein. This indicatesthat interactionsbetweentheseproteinsare not strong enoughto producesignificant changesin the enthalpyof denaturation. CONCLUSIONS
T 0.02 J/set
-!I
20
40
60
80
TEMPERATURE
(‘cl
120
100
Fig. 6- DiiYerential scanning calorimetry thermograms of 100% iron-saturated lactoferrin (a), 30% iron-saturated lactoferrin lb), apolactoferrin (cl, f3lactoglobulin (d) and a mixture of plactoglobulin and 100% iron-saturated lactoferrin (e). The amount of lactoferrin or p-lactoglobulin was 1.2 mg per pan in phosphate buffer.
Table 4- Denaturation temperatures and enthalpies of denaturation for lactoferrin with different degrees of iron saturation, for P/actoglobulin and for a mixture of @lactogiobulin and 100% iron-saturated lactiferrin AU -.AH (kJ.mol-I) To (“C) -.‘I 1 Protein (J.gApolactoferrin Lactoferrin sat.tO% Lactoferrin
s&.100%
p-Lactoglobulin (p-Lg) Mixture p-Lg + Lf (1:l by weight)
(Lfl
60.6 69.6 66 74 86.5 73.0 74.3 86.6
6.6 17.6
664 1,410
21 .o
1,600 127
The thermogramthat correspondsto 30% iron-saturatedlactoferrin showeda major peak with a temperatureof denaturation of 69.6”C. Therewas alsoa small peakwith a temperature of denaturationequal to the secondpeak of the 100% ironsaturatedthermogram.Apolactoferrin subjectedto DSC gave single endotherm,which suggestedthat the two lobeswithout 878-JOURNAL
OF FOOD SCIENCE-Volume
57, No. 4, 1992
THE DENATURATION of lactoferrin, with or without bound iron is slower when heat-treatedin phosphatebuffer than in milk. This differencemay be due to the decreasein pH of milk observedwith increasingtemperature,and also to the interactions of lactoferrin with caseinsand some whey proteins. However, presenceof P-lactoglobulindid not seemto notably affect denaturationwhen analyzedby calorimetry.The stability of lactoferrin to heat treatmentincreasedgreatly with iron saturation suggestingthat the two lobes of lactoferrin presenta different thermoresistance.The kinetic values for lactoferrin denaturtionpermit calculationsof the thermoresistanceof this proteinat differenttemperatures andtreatmenttimes.The widely usedpasteurizationtreatmentat 72-74°C for 15 set had practically no effect on lactoferrin structure. However, the evaporation treatmentat high temperatureappliedto’many formula milks could denaturethe major part of lactoferrin. Thus, supplementationwith lactoferrin after treatmentshouldbe considered in order to retain its biological functions. REFERENCES Aisen, P. and Liatowsky, I. 1980. Iron transport and storage proteins. Arm. Rev. Biochem. 49: 357. Anderson, B.F., Baker, H.M., Dodson, E.J., Norris, G.E., RumbaIl, S.V., Wat rs, J.M., and Baker, E.W. 1987. Structure of human lactoferrin at 3.2 .t resolution. Proc. Natl. Acad. Sci. USA 84: 1769. Anderson, B.F., Baker, H.M., Norris, G.E., Rice, D.W., and Baker, E.N. 1989. Structure of hum n lactoferrin crystallographic structure analysis and refinement at 2.8 x resolution. J. Mol. Biol. 209: 711. Anderson, B.F. 1990. A olactoferrin structure demonstrates Iigand-induced conformational cRange in transfer&s. Nature 344: 784. Baker, E.N., RumbaIl, S.V., and Anderson, B.F. 1987. Transferrina insights into structure and function from studies on lactoferrin. Trenda Biochem. Sci. 12: 350. Brock, J.H. 1985. Transferrins. In Metdlopmteina, Part 2. p. 183. McMilIan, London. Broxme er, H.E., Gentile, R., Cooper, F.S., Lu, L., Julianq, L., Piacibello, WJ eyers, P.A., and Cavanna, F. 1984. Functional activities of acidic isdferritins and lactoferrin in vitro and in vivo. Blood Cells 10: 397. Condbn,. S., Mpez, P., Oria, R., and SaIa, F.J. 1989, Thermal death deter~;;y design and evaluation of a thennoresmtometer. J. Food l&n. Con,’TM Mazurier J S ik G Montreuil J and Peters T.J. 1979 Iron-binding proteins in 8. in!& of iron acr&s’ihe duodenal brush bar: der. Evidence for specific lactotransferrin receptors in the human intestine. Biochim. Biophys. Acta 588: 120. DaIgleish D.G. 1990. Denaturation and a caseins in heated miIk. J. Agric. Food &%!a%n&?um proteins and Dannenber F. and Kessler, H.G. 1986. Reaction kinetics of the denaturation of w% ey proteins in milk. J. Food Sci. 63: 256. Davidson, L.A. and Liinnerdal, B. 1988. Specific binding of lactoferrin to
brush-border membrane: onotgeny and effect of glycan chain. Am. J. Physiol. 254: G580. Davidson, L.A. and tinnerdal, B. 1989. Fe-saturation and proteolysis of human lactoferrin: effect on brush-border receptor-mediated uptake of Fe and Mn. Am. J. Physiol. 257: G930. Evans, R.W. and Williams, J. 1978. Studies of the binding of different iron donors to human serum transferrin and isolation of iron-bindine fraements from the N- and C-terminal regions of the protein. Biocgm. 3. 173: 543. Ford, J.E., Law, B.A., Marshall, V.M.E., and Reiter, B. 1977. Influence of the heat treatment of human milk on some if its protective constituents. J. Pediatr. 90: 29. Fransson, G.B., Thoren-Tolling, K., Jones,. B., Hambraeus, L., and I&rnerdal, B. 1983a Absorption of lactoferrm iron in suckhng pigs. Nutr. Res. 3: 373. Frans~on~ G.B., Keen, C.L., and I.&merdal, B. 1983b. Supplementation of milk wrth iron bound to lactoferrin using weanling mice: I. Effects on hematology and tissue iron. J. Pediatr. Gastroenterol. Nutr. 2: 693. Fuchs, S. and Sela, M. 1986. Immunoadsorbents. Handbook of Experimental Immunology, Blackwell Scientific Publications, oxford. Goldblum, R.M., Dill, C.W., Albrecht, T.B., Alford, E.S., Garza, C., and Goldman, A-S. 1984. Rapid high-temperature treatment of human milk. J. Pediatr. 104: 380. Harwalkar. V.R. 1980a. Measurement of thermal denaturation of C-lactoglobulin at H 2.5. J. Dairy Sci. 63: 1043. Hanvalkar,V. i . 1980b. Kinetics of thermal denaturation of 8-lactoglobulin at pH 2.5. J. Dairy Sci. 63: 1052. Hekman, A. 1971. Association of lactoferrin with other proteins, as demonstrated by changes in electrophoretic mobility. Biochim. Biophys. Acta 251: 380. Houghton, M.R., Gracey, M., Burke, V., Botrell, C., and Spargo, R.M. 1985. Breast milk lactoferrm levels in relation to maternal nutritional status. J. Pediatr. Gestroenterol. 4: 230. Hu, W.L., Masurier, J., MontreuiI, J., and Spik, G. 1990. Isolation and partial characterisatron of a lactotransferrin receptor from mouse intestinal brush border. Biochemistry 29: 535. Jorieux, S., Mazurier, J., Montreuil,. J. and Spik, G. 1985. Characterisati.o,” of lactotransferrin complexes m human milk. Prot. Biol. Fluids 32:
Masson, P.L. and Heremans, J.F. 1971. Lactoferrin in milk from d&rent
629: 399. Miyauchi, J. and Watanabe, Y. 1987. Immuno@cchemical localization of lactoferrin in human neutrophils. Cell Tiss. Res. 247: 249. Moreau, M.C., Duval-Blah, Y., Muller, M.C., Raibaud, P., Vial, M., Gabilan. J.C.. and Daniel, N. 1983. Effet de la lactoferrine bovine et des Ig G bovines ~donnCa per.08 sur l’implantation de Escherichiu coli dane 16 tube digestif de sour-is gnotox&iques et de nouveau-n& humains. Ann. Microbial. 134B: 429. Nichols, B.L., McKee, KS., Henry, J.F., and Putman, M. 1987. Human lactoferrin stimulates thvmidine incorooration into DNA of rat crvnt .. . cells, Pediatr. Res. 21: 663. Nichols, B.L., McKee, KS., Putman, M., Henry, J.F., and Nichols, V.N. 1989. Human lactoferrin suunlementation of infant formulas increases thymidine incorporation in& the DNA of rat uypt cells. J. Pediatr. Gastroenterol. Nutr. 8: 102. Paulsson, M. and Dejmek, P. 1990. Thermal denaturation of wh ogt$s . in mixtures with caseins studied by differential scanning CZK J. Deiry Sci. 73: 590. Riie M., Moor! U., and Blanc, B. 1977. A calorimetric study of the ther-rn2. denaturatron ,. .^.. of whey proteins in simulated milk ultraSItrat-e. J. IJalry Kes. 44: OUY. Sanchez, L.. Aranda. P.. P&z. M.D., and Calvo, M. 1988. Concentration of la&fe&in and transfer&r throughout la&ion in cow’s colostrum .and milk. Biol. Chem. Hoppe-Seyler 369: 1005. V~~~~~~~a~~~~~~~~~~~~~~ &f3&1;+37$ 351. Walstra, P. and Jenness, R. 1984. Dairy Chemistry and Physics. John Wiley and Sons, New York. Watanabe, T., Nagura, H., Watanabe,-K., and Brow?, W.R. 1984. The it8dmf30f human nnlk lactoferrm to unmunoglobuhn A. FEBS Letters MS r&eiv&l 12/4/91; revised I/16/92; accepted 2/11/92.
This work WBB supported by pant and Dr. Sala.Trapat for collaboration for helpful sugge&ions.
ALI 90.0363 from CICYT. We thank Dr. CaxbSn with the thermoreaistometer and also Dr. Bmck
NMR DETECTION OF CHOLESTEROL OXIDE. . . From page 872 to conventionalcholesterolanalysis.The methodoffers high specificity and good sensitivity. It leadsto direct and unequivocalcharacterization of theunderivatized COx by NMR analysis with no chemicaltreatment.Moreover,the methodclearly revealspossibleco-occurrence of compounds with the sameHPLC retentiontime. The importanceof this methodis not limited to quantitativedetectionof the COx. The use of high-field NMR spectrometers,rathercostly for routine analyses,offers great potential for the objective study of intermediatesand productsderiving from cholesterolthroughchemicaltransformation during storageor heating. Studiesarenow in progressapplyingthis new methodology to quantitativeanalysisof the COx in fresh as well as aged commercialfoodstuffs. REFERENCES Addis P.B. 1986. Occurrence of lipid oxidation products in Food. Food Chem. Toxicol. 24(10/111: 1021-1030. Ansari GAS., Walker R.D., Smart V.B., V.I and Smith L.L. 1982. 182. Further investigations of mutagenic cholesterol preparations. Food Chem. Toxicol. 20: 35. Belits H.D. and Grosch W. 1987. Food Chemistry, p. 411414, SpringerB; Verlag, Berlin and Heidelberg. Cleveland M.Z. and H s N.D. 1987. Oxidation of cholesterol in commercially merciallv processed Drocessedco 7 s milk. J. Food Prot. 60: 867. Finoccl Finocchiaro E.T. and Richardson T. 1983. Sterol oxides in foodstuffs: A review. J. Food Prot. 46: 917. Hurst W.J., Aleo M.D., and Martin R.A. Jr. 1986. HPLC determination of the cholesterol content of egg noodles as an indicator of egg solids. J. Agric. Food Chem. 33: 820. Imm H., Werthessen N.T., Taylor C.B., and Lee K. 1976. Anglotoxicity and arteriosclerosis due to contaminants of USP-grade cholesterol. Arch. Pathol. Lab. Med. 100: 565. Kandutsch AA and Chen H.W. 1973. Inhibition of sterol s thesis in cultured mouee cella by 7a-h xycholesterol, 78hydroxycho f esterol and 7-ketocholesterol. J. Biol. C I?-em. 248: 8408. Maerker G. 1987. Cholesterol autoxidation-Current Status. JAOCS 64: Milker G. and Brunick F.J. 1986. Measurement of the 5 G-epoxides during cholesterol oxidation in aqueous dispersion. JAOCS’63: 771.
Morgan J.N. and Armstrong D.J. 1987. Formation of cholesterol-5,6-epoxides during spray-drying of egg yolk. J. Food Sci. 62: 1224. Morgan J.N. and Armstrong D.J. 1989. Wide-bore capillary gas chmmatographic method for quantilication of cholesterol oxidation products in egg yolk powder. J. Food Sci. 64: 427. Nourooz-Zadeh J. and Ap lqvist LA. 1988a. Cholesterol oxides in Swedish foods and food ingr eiYents: Milk powder products. J. Food Sci. 63: 74. Nouroos-Zadeh J. and Appelqvist LA. 1988b. Cholesterol oxides in Swedish foods and food ingredients: Butter and cheese. JAOCS 65: 1635. Park S.W. and A@is P.B, I986a. Capillary column gas-liquid chromatof7aphrc resolutron of ox&red cholesterol derrvatrves. Anal. Chem. 149: Park’S.W. and Addis P.B. 1985b. HPLC determination of C-7 oxidised cholesterol derivatives in foods. J. Food Sci. 50: 1437. Pen S.K Taylor C.B Tham P Werthessen N.T and Mikkelson B. 1978. Et%& Gf auto-oxid&on rod&s from cholest&l on aortic smooth muscle cells. Arch% Path. La% , Med. 102: 57. Pie E.J., S K, and Seillan C. 1990. Evaluation of oxidative degradation oP cholesterol in food and food ingredients: identification and quantillcation of cholesterol oxides. J. Agnc. Food Chem. 38: 973. Sander B.D., Addis P.B.? Won Park S., and Smith D.E. 1989a. Quantiflr2trg;f cholesterol oxldatron products m a vanety of foods. J. Food Pmt. Sander g D Smith DE Addis P B and Park SW 1989b Effects of prolonged &rd adverse s&age co&ions on levels of cholesterol oxidation products in deiry products. J. Food Sci. 64: 874. Sevaman A. and Peterson A-R. 1986. The cytotoxic and mutagenic roperties of cholesterol oxidation products. Food Chem. Toxic. 24: 110Fi. Still C.W., Kahn M., and -M&a A 1978. Rapid chromate a hit technique foo2reparatrve separations wrth moderate resolution. $? 8rg. Chem. 43: Sugino K. Terao J Murakami H., and Matsushita S. 1986. Hi h-performance liq&d chro$atography method for the quantification of cholesterol epoxides in spray-dried J. Agric. Food Chem. 34: 36. P erthessen N.T., Tham P., and Lee KT. 1979. ng angiotoxic derivatives of cholesterol. Am. J. Tsai L.S. and Hudson CA 1986. Cholesterol oxides in commercial dry egg products: Quantitation. J. Food Sci. 60: 229. Tsai L.S., Bichi K., Hudson C.A. and Meehan J.J. 1987. A method for the quantitative estimation of cholesterol a-oxide in e Lipids 15: 124. MS received g/30/91; revised 12/14/91; accepted l/10 F92.
We are very ~atefol to Prof. F. Addeo for valuable to Miss D. Ricziardi and Mr. R. Turbo for technical
diemeaiom. contrihutionn.
Thanks are extanded The work WBB sup
ported by the strat.cgicCNR project “QusIiti e tipicite degli Alimenti: metodologia innovativa di indaelne.”
Volume 57, No. 4, 1992-JOURNAL
OF FOOD SCIENCE-879
