Journal of Dairy Research (1997) 64 221230

Printed in Great Britain

221

Application of capillary electrophoresis to the study of proteolysis of

caseins
B ISIDRA RECIO, LOURDES AMIGO, MERCEDES RAMOS
ROSINA LOPEZ-FANDINN O
Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3,
E-28006 Madrid, Espanh a
(Received 2 May 1996 and accepted for publication 23 September 1996)
S. Capillary electrophoresis using a hydrophilically coated capillary and a
low pH buffer containing urea has been used to follow the proteolytic action of
plasmin and chymosin on isolated casein fractions, whole casein and individual milk
samples selected on the basis of their genetic variants. Several of the main casein
breakdown products were identified. These included, among others, -casein
"
(CN) A", -CN A#, -CN B, -CN C, -CN A, -CN A$, -CN B, -CN A and
"
"
"
#
"
#
$
-CN B, as well as proteose peptones, arising from the action of plasmin on the
$
different genetic variants of -CN. s -I-CN and s -CN f(1-23) from s -CN, and
"
"
"
para--CN and caseinomacropeptide from -CN produced by chymosin action were
also separated. The knowledge of their migration times provided information on the
extent and origin of casein hydrolysis in both milk and cheese, as found in samples
of proteolysed milk or fresh cheese.
Capillary electrophoresis (CE) is a fast growing technique that is finding new
applications in the analysis of milk proteins and peptides owing to its well known
advantages over electrophoretic and chromatographic methods. CE techniques
employ narrow bore capillaries (20200 m i.d.) to perform high efficiency
separations of both large and small molecules. These separations are facilitated by
the use of high voltages, which may generate electro-osmotic and electrophoretic
flow of buffer solutions and ionic species.
CE at basic pH with uncoated capillaries has been used for the separation and
quantification of whey proteins (Otte et al. 1994 ; Paterson et al. 1995 ; Recio et al.
1995). An uncoated capillary was also used for the analysis of caseinomacropeptide
from rennet whey (Otte et al. 1995). Van Riel & Olieman (1995) increased the
resolving power to 0510'1010' plate numbers}m by using a hydrophilically
coated capillary and applied it to the detection of rennet whey solids in skim milk
and acid buttermilk powder. De Jong et al. (1993) published a CE method using a
hydrophilically coated capillary that made possible the simultaneous separation of
whey proteins and caseins, including some genetic variants. The use of a coated
capillary in combination with a low pH buffer containing urea and methylhydroxyethyl cellulose avoided the undesirable adsorption of proteins on to the
capillary wall, usually associated with fused silica walls. Electro-osmotic flow (bulk
flow of liquid in the capillary as a consequence of the surface charge on the interior
capillary wall) was virtually zero under these conditions and the migration behaviour
of peptides and proteins depended only on their effective mobility at low pH. With

222

I. R

this method, theoretical plate numbers in the range 0310'0610' were obtained
(De Jong et al. 1993).
This method was recently optimized by Recio & Olieman (1996) in order to
provide a quantitative determination of the proteins separated in the electropherograms, and to detect the serum protein : casein ratio in different dairy products,
as well as the addition of milk powder to pasteurized milk. However, so far very few
studies have applied CE to the investigation of proteolysis in milk or cheese
(Kristiansen et al. 1994).
In an attempt to identify the CE pattern corresponding to the main degradation
products arising from the action of different proteolytic agents of importance in the
technology of dairy products, we have carried out a study on the action of plasmin
(EC 3\4\21\7) and chymosin (EC 3\4\23\4) on isolated casein fractions, whole
casein and individual milk samples selected on the basis of their genetic variants.

Samples
Isoelectric casein was prepared by precipitation from whole milk by adding 2 HCl to pH 46, followed by centrifugation at 4500 g for 15 min. The casein precipitate
was washed three times with 1 -sodium acetate buffer, pH 46. The remaining fat in
the casein precipitate was removed by washing with dichloromethanesodium
acetate buffer (1 : 1, v}v). The final casein precipitate was lyophilized. Casein
fractions (s-, - and -casein (CN)) were purchased from Sigma Chemical Co. (St
Louis, MO 63178, USA).
In order to identify the different genetic variants of -CN, individual milk
samples containing -CN A"A", A#A#, A#B, A"C and A"A$ variants were selected from
a pool of 631 samples. s -CN f(1-23) was a gift from Dr C. Mart! n Hernandez
"
(Instituto del Frio). Caseinomacropeptide was prepared by purification, after
successive injections on reversed-phase HPLC, of the fraction soluble in trichloroacetic acid (40 g}l) of a chymosin digest of isoelectric casein (Lo! pez-Fandin4 o et al.
1993).
Fresh cheeses (pH 594), manufactured from pasteurized cows milk (72 C, 15 s)
with the addition of suitable starter cultures (Lactococcus lactis subsp. lactis and Lc.
lactis subsp. cremoris), were purchased from a local market. Caseins from cheese were
obtained by precipitation at pH 46 and washed and lyophilized as above. Raw milk
samples were treated with thimerosal (01 g}l ; Scharlau, E-08016 Barcelona, Spain)
and incubated at 37 C for 48 h.
Preparation of model systems
Isoelectric casein and casein fractions were dissolved to 3 mg}ml in 01 potassium phosphate buffer, pH 65, except for the chymosin digests where the
concentration was 25 mg}ml. Proteolysis with plasmin and calf chymosin, both from
Sigma, were carried out at enzyme : substrate ratios of 16710$ and
47210' units}mg casein respectively. Individual milk samples were digested with
plasmin at an enzyme : substrate ratio of 510# units}ml.
Model systems were incubated at pH 65 and 37 C for different periods between
0 and 2 h. Portions were withdrawn from the mixtures at intervals, reactions stopped
by dilution at 1 : 15 with CE sample buffer, pH 8601 (see below), and the sample
injected without further preparation. In some cases, the pH of the reaction mixtures
was adjusted to 46, in order to separate products soluble and insoluble at this pH,
before the addition of CE sample buffer.

Capillary electrophoresis of casein

223

Capillary electrophoresis
CE buffers were prepared following the method of Recio & Olieman (1996).
Sample buffer (pH 8601) consisted of 167 m-Tris(hydroxymethyl)aminomethane (reagent grade from Sigma), 42 m-3-morpholino-propanesulphonic
acid (BioChemika MicroSelect ; Fluka, CH-9470 Buchs, Switzerland), 67 methylenedinitrilotetra-acetic acid disodium salt dihydrate (Tritiplex III ; Merck, D64293 Darmstadt 1, Germany), 17 m--dithiothreitol (Sigma), 6 -urea(Sigma)
and methylhydroxyethyl cellulose (05 g}l, 30 000 ; Serva, D-69042 Heidelberg 1,
Germany).
The electrophoresis buffer was 032 -citric acid20 m-sodium citrate6 -urea,
pH 3001 containing 05 g methylhydroxyethyl cellulose}l. Before use, buffers
were filtered through a 022 m filter (Sterile Acrodisc2 with HT Tuffryn membrane ;
Gelman Sciences, Ann Arbor, MI 48106, USA).
CE was carried out using a Beckman P}ACE System 2050 controlled by a System
Gold Software data system version 810 (Beckman Instruments Inc., San Ramon, CA
94583-0701, USA). The separations were performed using a hydrophilic-coated
fused-silica capillary column (CElect P1 ; Supelco, Bellefonte, PA 16823, USA) of
047 or 057 m50 m i.d., with a slit opening of 100800 m. Separations were as
described by Recio & Olieman (1996) with final voltages of 20 and 25 kV respectively
for the 047 and 057 m capillaries. Detection was performed on the column at
214 nm. In the electropherograms, absorbance is represented against the time
required for a solute to migrate from the beginning of the capillary to the detector
window. When using the same capillary and running buffer, the relative for
migration times were ! 008 %. However, changes in the migration times due to the
use of new capillaries or buffer solutions have been observed. In addition, small
variations were noted when analysing casein fractions as compared with milk
samples, probably caused by the presence of different ions in the sample matrix. For
this reason, identification of milk proteins and peptides was achieved by spiking,
whenever standards were available, or by considering relative migration times.
For the purposes of identification, in addition to the CE separations all the
samples were simultaneously analysed by alkaline-PAGE (Ramos et al. 1977), SDSPAGE (Laemmli, 1970) and isoelectric focusing (Bovenhuis & Verstege, 1989). Thus,
a full characterization of the hydrolysates by conventional separation techniques
was achieved.

Hydrolysis with plasmin
Fig. 1 shows the electropherograms of whole casein prior to hydrolysis, together
with whole casein, - and s-CN treated with plasmin. Certain incubation times have
been selected in order to show the most representative breakdown products arising
from the action of the enzyme. As expected, the main degradation products resulted
from the action of the enzyme on -CN (Fig. 1 c ; Grufferty & Fox, 1988). These
included various peaks belonging to the -CN, as well as four other peaks that
probably corresponded to hydrophilic proteose peptone components (Andrews &
Alichanidis, 1983), as they were the only degradation products from the hydrolysate
that remained soluble at pH 46 (marked S in Fig. 1 b, c).
Owing to its high content of lysyl residues, s -CN has numerous plasmin#
sensitive bonds (Le Bars & Gripon, 1989). Indeed, s -CN also was rapidly cleaved
#
by plasmin, and disappeared from the hydrolysates of s-CN and whole casein after

I. R

224
0020
(a)

s1

0015

bA1

0010

s0
s2

0005

0020

bB

s0

s1

bA2

s2

0010

sP

bA1

0005
A 214

(b)

0015

0020

bA2

c c
S

(c)

0015
0010

bA2
bB bA1
c
cc c
S S

0005
0

(d)
0020

s1

0015

0010

sP

s0

0005
0
10

20

30

40

Time, min
Fig. 1. Electropherograms of different protein fractions digested with plasmin, with incubation times
in parentheses : (a) whole casein (0 min), (b) whole casein (20 min), (c) -casein (CN) (10 min), (d) sCN (60 min). Separation was in a hydrophilically coated fused silica capillary (047 m50 m, 040 m
to detection point), temperature 45 C, injection 15 s, linear voltage gradient 020 kV in 3 min,
followed by constant voltage of 20 kV, separation buffer 6 -urea032 -citric acid20 m-sodium
citrate, pH 30 containing 05 g methylhydroxyethyl cellulose}l. , -CN ; s , s -CN ; s -, s -CN ;
" "
!
!
sP, peptide derived from the action of plasmin on s -CN ; B, -CN B ; A", -CN A" ; A#, -CN A# ;
"
, -CN ; S, peptides soluble at pH 46 arising from the action of plasmin on s-CN and -CN.

225

Capillary electrophoresis of casein

bA2

(a)
004
s1
003
b-Lg

002

-La

A 214

001

s0

s22

(b)

bA1

004

003

002
2
5
001

0
10

S
S

20

30

40

Time, min
Fig. 2. Electropherograms of individual milk samples digested with plasmin for 20 min : (a) milk
containing -casein (CN) A#A#, (b) milk containing -CN A"A". -La, -lactalbumin ; -Lg, lactoglobulin ; s , s -CN ; s , s -CN ; s , s -CN ; A#, -CN A# ; A", -CN A" ; , -CN ; S, peptides
# #
" "
! !
soluble at pH 46 arising from the action of plasmin on s-CN and -CN. 1, sP, peptide derived from
the action of plasmin on s -CN ; 2, -CN A ; 3, -CN A# ; 4, -CN A ; 5, -CN A". Electrophoretic
"
#
"
$
"
conditions were as in Fig. 1.

15 and 20 min treatment respectively. The electrophoretic pattern of s-CN following

plasmin treatment showed a fast migrating degradation product which was also
observed in the hydrolysate of whole casein (sP in Fig. 1 b, d). It is likely that this
peptide was produced from s -CN, which was attacked more slowly, as it was still
"
being released well after all the s -CN had been completely hydrolysed by the
#
enzyme. In addition, there were some slow moving peptides resulting from the action
of plasmin on s-CN, which were also present in the hydrolysate of whole casein. One
of those (marked S in Fig. 1 d) remained soluble at pH 46. Plasmin acts on s -CN
"
to yield a heterogeneous group of peptides termed -CN (Aimutis & Eigel, 1982).
Recently, Addeo et al. (1995) isolated the peptide s -CN f(80-199), which derived
"
from the degradation of s -CN by plasmin.
"
Plasmin hydrolysates of milks containing -CN A"A" and -CN A#A# enabled us
to identify -CN A", -CN A#, -CN and -CN (Eigel et al. 1984) as illustrated in
"
"
#
$
Fig. 2. When using a hydrophilically coated capillary, the electro-osmotic flow is
suppressed and the electrophoretic migration of proteins depends on their mass and
charge characteristics at pH 30 (De Jong et al. 1993). Because of its higher positive

I. R

226
003

s1
(a)
b B bA2

002

b-Lg
-La

001

s2
6 2
78

0
003

s0
3

(b)

A 214

b C bA1
002

2
4

001
9

5
S

(c )
s1BC
b A1 b A3
s0BC

002

11 10
2+
4
5

001

0
10

20

30

40

Time, min
Fig. 3. Electropherograms of individual milk samples digested with plasmin for 20 min : (a) milk
containing -casein (CN) A#B, (b) milk containing -CN A"C, (c) milk containing -CN A"A$. -La,
-lactalbumin ; -Lg, -lactoglobulin ; s , s -CN ; s , s -CN ; s BC, s -CN BC ; s BC, s -CN BC ;
" "
! !
"
"
!
!
A#, -CN A# ; B, -CN B ; A", -CN A" ; C, -CN C ; A$, -CN A$ ; , -CN ; S, peptides soluble at
pH 46 arising from the action of plasmin on s-CN and -CN. 1, s-P, peptide derived from the action
of plasmin on s -CN ; 2, -CN A ; 3, -CN A# ; 4, -CN A ; 5, -CN A" ; 6, -CN B ; 7, -CN B ; 8,
"
#
"
$
"
#
"
-CN B ; 9, -CN C ; 10, -CN A$ ; 11, -CN A$. Electrophoretic conditions were as in Fig. 1.

"

"

charge, -CN had the lowest migration time, followed by -CN A" (Fig. 2 b) and #
"
"
CN A# (Fig. 2 a). (In this case the substitution of His'( in -CN A" for Pro'( in -CN
A# made it possible to separate them at acidic pH.) -CN had the longest migration
$
time.
The electrophoretic patterns resulting from the action of plasmin on milks
containing -CN A#B, -CN A"C and -CN A"A$ variants are shown in Fig. 3. The
substitution of Ser"## in -CN A for Arg"## in -CN B, in addition to the presence of
His'(, as in -CN A" (Eigel et al. 1984), rendered -CN B, -CN B and -CN B more
#
$
"
electropositive than their variant A counterparts (Fig. 3 a). Similarly, the

227

Capillary electrophoresis of casein

s1

005

bA2

(a)
bA1

004
003

s0

p-

002

s
f(1-23)

s2

s1-I
s1-X

001

CMP

0
(b)
012

A214

010
008
p-

006
004

CMP

002
0
(c )

s1
s0

004
003
002

s1-I
s1
f(1-23)

s2

s1-X

001
0
10

20

30

40

Time, min
Fig. 4. Electropherograms corresponding to different protein fractions digested with chymosin, with
incubation times in parentheses : (a) whole casein (10 min), (b) -casein (CN) (10 min), (c) s-CN
(120 min). s , s -CN ; s , s -CN ; s , s -CN ; A", -CN A" ; A#, -CN A# ; p-, para--CN ; CMP,
# #
" "
! !
caseinomacropeptide ; s -I, s -I-CN ; s -X, peptide derived from s-CN ; s f(1-23), s -CN f(1-23).
"
"
"
"
"
Electrophoretic conditions were as in Fig. 1.

substitutions of SerP$& and Glu$( in -CN A" for Ser$& and Lys$( in -CN C, together
with the presence of His'( (Eigel et al. 1984), contributed to the high positive charge
of -CN C and thus to its low migration time (Fig. 3 b). -CN A$ was tentatively
"
"
identified in view of the amino acid substitutions Pro'( and Gln"!' of -CN A$, which
make it less electropositive than -CN A" and -CN A# (Fig. 3 c). However, -CN
"
"
#
A$, which should appear as a peak with lower migration time than -CN A" by virtue
#
of the substitution of His"!' for Gln"!', was not found in the plasmin digest of milk
containing -CN A"A$, maybe owing to coelution with -CN A". The presence of s "
"
CN BC and s -CN BC in the electropherogram illustrated in Fig. 3 (c) should be
!
noted. Trieu-Cuot & Gripon (1982) separated -CN A" and -CN A# by isoelectric

I. R

228
008

s1

(a )

A214

006

bA2
bA1
s0

004
-Lg
-La
002

s2

1
5
0
10

S
S

20

30

Time, min

007

(b )

s1

006

bA2
bA1

005
s0

A214

004
p-
003
002
001

-Lg s2 2
7
5
-La
6

bB
4
S

S -I
s1
S

0
001
10

20

30

40

Time, min
Fig. 5. Electropherograms corresponding to (a) milk incubated for 48 h at 37 C in the presence of 01 g
thimerosal}l (electrophoretic conditions were as in Fig. 1), (b) casein from a fresh cheese clotted
enzymically (electrophoretic conditions were as in Fig. 1 except that the total length of the capillary
was 057 m, and the final voltage 25 kV). -La, -lactalbumin ; -Lg, -lactoglobulin ; s , s -casein
# #
(CN) ; s , s -CN ; s , s -CN ; s -I, s -I-CN ; B, -CN B ; A", -CN A" ; A#, -CN A# ; , -CN ;
" "
! !
"
"
p-, para--CN ; S, peptides soluble at pH 46 arising from the action of plasmin on s-CN and -CN. 1,
sP, peptide derived from the action of plasmin on s -CN ; 2, -CN A ; 3, -CN A# ; 4, -CN A ; 5,
"
#
"
$
-CN A" ; 6, -CN C ; 7, -CN B.

"

"

Capillary electrophoresis of casein

229

focusing, as well as -CN A", -CN A#, -CN and -CN. However, to
"
"
#
$
our knowledge the separation of the degradation products of -CN A$, -CN B and
-CN C has not so far been achieved by conventional electrophoresis.
Hydrolysis by chymosin
The electrophoretic pattern of whole casein hydrolysed with chymosin is
illustrated in Fig. 4 (a). The main degradation products included para--CN and
caseinomacropeptide (identified by spiking with the purified peptide), resulting from
the action of the enzyme on the Phe"!&Met"!' bond of -CN, which is the primary
cleavage site of chymosin action and leads to the enzymic coagulation of milk (Fig.
4 b ; Dalgleish, 1987).
s -CN was also hydrolysed rapidly by chymosin at the Phe#$Phe#% bond, giving
"
rise to s -I-CN (s -CN f(24-199)) and s -CN f(1-23) (Fig. 4 c). This is one of the most
"
"
"
important events that takes place during the early stages of ripening of most cheese
varieties and is responsible for the softening of cheese texture (Creamer & Olson,
1982). s -I-CN is further hydrolysed by chymosin during cheese ripening, giving rise
"
to degradation products of high electrophoretic mobility in urea-PAGE at alkaline
pH (Grappin et al. 1985 ; McSweeney et al. 1993). The electropherograms of s-CN and
whole casein treated with chymosin (Fig. 4 a, c) showed peaks of longer migration
time than s -I-CN, which were produced at a rate similar to the hydrolysis of s "
"
I-CN.
Proteolysis in milk and cheese
Identification of the degradation products arising from different enzymes makes
this method suitable for studying proteolysis in milk and cheese, as illustrated in Fig.
5. The main breakdown products arising from plasmin action were observed in the
electropherogram of a sample of raw milk that had been kept at 37 C for 48 h in the
presence of bacterial inhibitors to promote protein degradation by native proteinases
(Fig. 5 a). The presence of para--CN, which might have been formed by the action
of residual psychrotroph enzymes, cannot be excluded as it co-migrates with lactoglobulin.
Fig. 5 (b) shows the casein fraction of a fresh cheese sample made from pasteurized
milk. Heat-denatured whey proteins can be observed in the electropherogram (Recio
& Olieman, 1996), with the two genetic variants of -lactoglobulin appearing as a
double shoulder on the para--CN peak. In addition to para--CN, the main
degradation products were -CN, s -I-CN and proteose peptone components.
"
The present results showed that capillary electrophoresis could be an efficient tool
for following the hydrolysis of caseins caused by various proteinases in milk and
cheese. This method provided a very high resolution of proteins and peptides
differing in just one amino acid substitution and, unlike conventional electrophoresis, there is no limitation in the size of the components to be separated. Several
of the main casein breakdown products have been identified.
The authors thank Ms C. Talavera for skilful technical assistance. This work has
been supported by project CAM COR0035}94.

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230

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