of casein and casein subunits
`analysis
`proteins:
`Human-milk
`by anion-exchange
`chromatography,
`gel electrophoresis,
`and specific
`staining methods13
`
`Clemens
`
`Kunz
`
`and Bo L#{246}nnerdal
`
`to
`
`in
`
`by
`
`functions
`biological
`several
`serve
`is believed
`milk
`human
`in
`Casein
`ABSTRACT
`so far
`received
`casein
`has
`ofhuman
`subunit
`composition
`and
`content
`the
`However,
`newborns.
`and
`casein
`human-milk
`whey
`a method
`to separate
`recently
`developed
`little
`attention.
`We
`human
`milk
`to pH 4.3 and
`addition
`ofcalcium
`followed
`by ultracentrifu-
`adjustment
`ofwhole
`In this
`study we analyzed
`and evaluated
`human
`casein
`prepared
`by different
`methods.
`gation.
`We used
`fast protein
`liquid
`chromatography
`(FPLC)
`with an anion-exchange
`column
`(Mono-
`Q) and
`polyacrylamide
`gradient
`gel
`electrophoresis
`techniques
`to analyze
`casein
`subunit
`the
`composition.
`Total
`in human
`milk,
`as determined
`by the Kjeldahl
`method,
`varies
`dur-
`ing
`lactation;
`the
`content
`is “-20%
`of
`the
`total
`protein
`content
`in early
`lactation
`and
`45% in late
`lactation.
`found
`differences
`in both
`glycosylation
`and
`phosphorylation
`patterns
`Am J Gun Nuir
`of K-caseins
`and
`from
`premature
`and
`term
`milk
`samples.
`1990;5
`1:37-46
`
`casein
`casein
`We
`/3-caseins
`
`KEY WORDS
`anion-exchange
`
`Human-milk
`chromatography,
`
`casein,
`proteins,
`gel
`electrophoresis
`
`K-casein,
`
`$-casein,
`
`casein
`
`subunits,
`
`FPLC
`
`Introduction
`
`the
`
`un-
`least
`represent
`subunits
`its
`and
`casein
`Human
`human
`in
`of proteins
`class
`and
`characterized
`derstood
`to micelle
`with
`regard
`is known
`example,
`little
`for
`milk;
`in phosphorylation
`variations
`submicelle
`assembly,
`and
`total
`or
`the
`of
`glycosylation
`of
`or
`(cid:1)3-casein
`K-casein,
`funda-
`In addition,
`the
`milk.
`amount
`in human
`ofcasein
`to physiological
`functions
`mental
`with
`regard
`questions
`and
`casein
`fragments
`need
`of
`casein,
`subunits,
`casein
`(1).
`There
`are
`some
`reports
`considerable
`study
`further
`influence
`the
`intestinal
`that
`casein
`phosphopeptides
`ab-
`and
`that
`these
`peptides
`may
`sorption
`calcium
`(2-4)
`of
`have
`a regulatory
`role
`in the mineralization
`process
`of
`calcified
`tissues
`5). The
`complex
`carbohydrate
`(2,
`position
`(6-8)
`functions
`of human
`ic-casein
`and
`be
`investigated
`further.
`The
`discovery
`of
`casein
`ments
`with
`opioid-like
`activities
`(9-12)
`immunostim-
`or
`ulatory
`properties(13-l
`5) may
`unravel
`additional
`physi-
`obogical
`functions
`of casein.
`available
`There
`is currently
`no method
`and
`distinct
`separation
`ofwhey
`proteins
`currently
`used were
`developed
`primarily
`which
`is
`in
`several
`aspects
`Vine
`casein,
`from human
`casein.
`The
`differences
`in the
`uble whey
`proteins
`in human
`and
`bovine
`the marked
`differences
`in the
`content
`subunits
`between
`the
`two
`species
`makes
`methods
`for
`analyzing
`bovine
`casein,
`such
`
`com-
`to
`need
`frag-
`
`to a
`leads
`that
`casein. Methods
`to analyze
`bo-
`quite
`different
`content
`of sol-
`milk
`as well
`as
`in the
`casein
`it unlikely
`that
`as adjustment
`
`and
`
`to
`
`pH
`used
`
`of
`problem
`are based
`nitrogen
`the
`
`by
`milk.
`
`the
`
`A
`
`con-
`
`can
`(16),
`centrifugation
`followed
`4.6
`of milk
`acid
`that
`It
`is
`known
`human
`for
`be
`easily
`various
`to
`casein
`leads
`human-milk
`of
`precipitation
`(1 7-20).
`whey
`proteins
`coprecipitating
`amounts
`casein
`on
`data
`literature
`is that
`further
`determining
`methods
`either
`upon
`indirect
`tent
`and
`calcu-
`and
`whey
`content
`ofwhole
`milk
`the
`gel
`electrophoresis
`amount
`casein
`or upon
`of
`lating
`indirect
`approach
`casein
`fractions.
`To use
`the
`ofwhole
`fractions
`with
`either
`to
`separate
`milk
`into
`is
`necessary
`electrophoresis
`Gel
`proteins
`caseins.
`or
`only
`whey
`and
`between
`to
`differentiate
`whole
`casein
`fl-casein
`K-
`in
`range
`reported
`because
`the
`appropriate,
`not
`does
`seem
`is 25 000-40
`(1 8, 21,
`000
`molecular
`weight
`for K-casein
`(cid:1)-24
`000
`22) whereas
`the molecular
`weight
`of(cid:1)3-casein
`is
`(23).
`It
`is therefore
`likely
`that
`there
`are
`some
`overlapping
`When
`acid
`precipitation,
`bands
`of
`/3- and
`K-casein.
`and
`the
`indirect
`approach
`(whole-milk
`mentation,
`N = casein
`gen
`- whey
`N)
`to
`analyze
`casein
`were
`
`it
`
`of
`
`sedi-
`nitro-
`corn-
`
`I From
`Davis.
`2 Supported
`
`the Department
`
`of Nutrition,
`
`University
`
`of California,
`
`by
`
`research
`
`contract
`
`NOl-HD-6-2923
`
`from
`
`the Na-
`
`of Health,
`
`and
`
`the Deutsche
`
`Forschungsgemeinschaft
`
`Institutes
`
`tional
`(CK).
`3 Address
`University
`Received
`Accepted
`
`to B L#{246}nnerdal,Department
`requests
`Davis,
`CA 95616.
`
`of Nutrition,
`
`reprint
`ofCalifornia,
`June
`6, 1988.
`for publication
`
`February
`
`23.
`
`1989.
`
`Am
`
`J C/in Nuir
`
`l990;5l:37-46.
`
`Printed
`
`in USA.
`
`© 1990
`
`American
`
`Society
`
`for Clinical
`
`Nutrition
`
`37
`
`
`1 of 10
`
`FRESENIUS-KABI, Exh. 1038
`
`

`
`38
`
`KUNZ
`
`AND
`
`LONNERDAL
`
`and
`within
`between
`conclusion
`apparent
`least
`in part,
`different
`the
`separation
`and
`milk
`after
`adjustment
`addition
`ofcalcium
`
`the methods
`is that
`the
`proteins.
`analysis
`ofthe
`followed
`
`of
`pH
`by
`
`The
`at
`
`large
`pared,
`obtained
`were
`three methods
`(cid:1)Recently,
`we
`whey
`proteins
`ofwhole
`milk
`ultracentrifugation
`In this
`paper
`to analyze
`casein
`chromatography.
`to SDS-polyacrylamide
`staining
`lowed
`by
`specific
`and
`purity
`of
`phosphorybated
`subunits.
`This
`method
`was
`from one mother
`during
`the
`changes
`in total
`casein
`gate
`tion
`and
`phosphorylation
`
`variations
`(19).
`detect,
`reported
`in human
`to 4.3 and
`(24).
`of a method
`development
`report
`we
`subunits
`by anion-exchange
`and
`casein
`The
`resulting
`fractions
`were
`subjected
`gradient
`gel
`electrophoresis
`fob-
`to
`demonstrate
`the
`identity
`and
`glycosybated
`casein
`applied
`to
`casein
`fractions
`lactation
`to investi-
`period
`content
`in the
`glycosyla-
`patterns
`casein
`subunits.
`
`the
`
`and
`ofthe
`
`Materials
`
`and methods
`
`at
`
`the
`
`Milk
`samples
`from the
`preparations
`casein
`the
`In this
`study we analyzed
`separa-
`we
`reported
`for which
`same
`human-milk
`samples
`samples
`proteins
`(24). Human-milk
`tion
`and
`analysis
`of whey
`of lactation.
`mothers
`at various
`stages
`were
`provided
`by healthy
`The
`protocol
`was
`approved
`by the Human
`Subjects
`Review
`Committee
`the University
`ofCalifornia,
`The
`samples
`Davis.
`of
`were
`collected
`at
`the
`second
`nursing
`the
`day
`by emptying
`one
`breast,
`gently
`shaking
`the
`individual
`milk
`samples,
`and
`taking
`an aliquot
`for analysis without
`prior
`freezing.
`in detail
`Preparation
`of human
`milk was
`done
`as described
`in our
`previous
`milk
`(24).
`paper
`Briefly,
`either
`whole
`human
`(4 mL)
`or skim milk was used
`fresh
`to test
`different
`variables.
`Samples
`were
`centrifuged
`at
`low speed
`(10 400
`x g)at
`4 #{176}Cwith
`or without
`pH adjustment(to
`pH 46)
`or at high
`speed
`(189
`000
`x g) at 4 or 20 #{176}Cwith
`or without
`pH adjustment
`(to pH 4.6 or
`4.3)with
`or without
`calcium
`(60 mmolCaClilL).
`After
`the
`final
`step,
`caseins
`were
`lyophilized
`fat and whey
`centrifugation
`fractions
`kept
`frozen
`-20
`#{176}Cuntil
`analysis.
`
`were
`
`at
`
`and
`further
`
`Anion-exchangefast
`
`protein
`
`liquid
`
`chromatography
`
`were
`on
`
`to fast
`HR
`
`liquid
`protein
`subjected
`samples
`casein
`Whole
`5/5
`anion-ex-
`a Mono-Q
`(FPLC)
`chromatography
`Piscataway,
`NJ).
`Fine
`Chemicals,
`(Pharmacia
`change
`column
`skim
`milk,
`the
`or
`human
`milk
`of whole
`After
`centrifugation
`ethanolamine/
`and
`dissolved
`in 20 mmol
`pellet was
`lyophilized
`L(pH 9.5, 4 mg casein/0.5
`mL). Solid
`added
`to a final
`urea
`was
`kept
`in a water-
`concentration
`of 6 mol/L.
`The
`samples
`were
`h. Then,
`j(cid:1)L
`bath
`(25 #{176}C)with occasional
`stirring
`for 0.5-1
`500
`room tempera-
`was
`injected
`onto
`the
`column
`and
`separated
`A, 20 mmol
`eth-
`ture
`by use ofthe
`following
`conditions.
`Buffer
`buffer
`B, buffer
`anolamine/L
`(pH 9.5) with
`6 mol
`urea/L,
`and
`fresh,
`filtered
`A containing
`0.6 mol NaCl/L,
`were
`prepared
`degassed
`and
`through
`0.22
`(cid:1)trn filters(Millipore,
`Bedford,
`MA),
`buffer A from 0 to
`3 mL
`and
`before
`use. Gradient
`A used
`gradient
`ofO-lOO%
`buffer
`B from
`3 to 30 mL
`in 27 mm.
`Gradi-
`ent B used
`buffer
`A from 0 to 3 mL and
`a gradient
`of 0-100%
`to
`buffer
`B from 3 to 57 mL in 54 mm. Gradient
`A was
`chosen
`identify
`whey
`proteins
`in the
`casein
`fraction;
`gradient
`B was
`used
`to differentiate
`between
`the
`casein
`subunits,
`fi- and K-ca-
`sein.
`The
`flow rate was
`1 mL/min,
`the
`recorder
`setting
`was
`0.5
`cm/mL,
`and
`the pressure
`1 .5 MPa.
`The
`column
`eluant
`was
`was
`monitored
`continuously
`at
`280
`nm.
`Linearity
`the
`ionic
`
`a
`
`at
`
`of
`
`by use of a con-
`checked
`regularly
`used was
`gradient
`strength
`Bridge,
`Fisher
`Scien-
`(Barnstead
`meter
`ductivity
`Conductivity
`identity
`and purity
`ofthe
`eluted
`frac-
`PA). The
`tific,
`Pittsburgh,
`tions
`were
`established
`by comparing
`the whole
`casein
`sample
`with
`the
`FPLC
`fractions
`by SDS
`polyacrylamide
`gradient
`gel
`electrophoresis
`(PAGGE).
`To identify
`the
`elution
`positions
`for
`whey
`proteins
`in the
`same
`buffer
`system,
`250
`(cid:1)L whey was
`di-
`loaded
`onto
`the column.
`luted
`250
`(cid:1)sL buffer
`with
`
`and
`
`gel
`
`electrophoresis
`Polyacrylamidegradient
`polyacryl-
`10-20%
`or
`in 3-27%
`performed
`was
`PAGGE
`gels;
`Integrated
`Sepa-
`(precasted
`containing
`1% SDS
`amide
`gels
`ration
`Systems,
`Newton,
`MA)
`as
`described
`(24)
`using
`the
`Hoefer
`Mighty
`Small
`Apparatus
`(Hoefer
`Scientific
`Instru-
`ments,
`Francisco,
`CA). Thelyophilized
`caseins(l
`mg)were
`San
`(cid:1)zL sample
`buffer
`(0.025
`mol Tris-HC1/L,
`dissolved
`in
`1000
`0.25% bromophenol
`blue,
`2.5% (cid:1)-mercap-
`0. 1% SDS,
`pH 6.8,
`mi-
`toethanol)
`and
`heated
`in boiling
`water
`1.5 mm.
`Fifteen
`for
`FPLC
`croliters
`ofthese
`solutions
`was
`loaded
`on the gel. Eluted
`by using
`anion-exchange-column
`fractions
`were
`concentrated
`Danvers,
`(Centricon
`tubes, Amicon,
`microconcentrator
`tubes
`MA).
`To
`40 (cid:1)zL Of
`zL sample
`concentrated
`fractions,
`60
`the
`heated,
`buffer was
`added,
`the
`sample
`was
`and
`15 (cid:1)L was
`loaded
`on the gel. Coomassie
`Blue
`staining
`and
`destaining
`were
`per-
`formed
`as described
`(24). Ethyl
`Stains
`All
`(Gallard-Schlesinger,
`Carle
`Place, NY) was used
`to differentially
`pure
`proteins,
`stain
`phosphoproteins,
`and
`acidic
`proteins
`in the
`same
`gel
`(25).
`milk
`To investigate
`the protein
`distribution
`ofwhole
`human
`by gel electrophoresis,
`40 (cid:1)sL milk
`(not
`delipidated)
`was diluted
`with
`160 (cid:1)L
`sample
`buffer
`and
`heated
`in boiling
`water
`for
`1.5
`mm,
`and
`10 (cid:1)L was
`loaded
`onto
`the
`gel. The
`relative
`distribu-
`tion
`of
`individual
`proteins
`after
`staining
`with Coomassie
`Blue
`was determined
`by densitometric
`scanning
`with
`a Hoefer
`OS
`Fran-
`300
`densitometer
`(Hoefer
`Scientific
`Instruments,
`San
`cisco,
`CA)
`using
`a 585-nm-wavelength
`filter.
`area
`under
`each peak was measured
`by integration.
`
`The
`
`Immunoelectrophoresis
`
`and
`
`immunodiffusion
`
`immunodiffusion
`radial
`and
`Immunoelectrophoresis
`in the
`supernatants
`proteins
`the whey
`whey
`fractions
`and
`performed
`according
`were
`was washed
`the
`casein
`pellet
`as described
`Laurell
`(26)
`and Mancini
`et al (27)
`(24).
`
`the
`of
`after
`to
`
`Protein
`
`content
`
`a modified
`with
`determined
`was
`content
`protein
`The
`in lyophilized
`protein
`content
`(28).
`The
`dahl method
`by multiplying
`the nitrogen
`content
`was determined
`The
`protein
`content
`in whey
`fractions
`was
`determined
`tracting
`nonprotein
`N from whey
`protein
`N multiplied
`
`Kjel-
`casein
`by 6.25.
`by sub-
`by 6.25.
`
`Carbohydrate
`
`analysis
`
`Neutral
`cording
`Dubois
`nerholrn
`
`sugars
`to Dische
`et al(30).
`(31).
`
`were
`(29)
`Sialic
`
`with
`quantified
`phenol
`and
`with
`acid was determined
`
`ac-
`reagent
`anthrone
`to
`according
`reagent
`according
`to Sven-
`
`Phosphorus
`analysis
`The
`phosphorus
`method
`ofEibl
`and
`
`Results
`
`content
`Lands
`
`was determined
`(32).
`
`according
`
`to the
`
`A distinct
`whey,
`and
`
`separation
`casein
`fractions
`
`milk
`human
`of whole
`by
`ubtracentrifugation
`
`into
`
`fat,
`with-
`
`
`2 of 10
`
`

`
`ANALYSIS
`
`L(cid:1)(cid:1))
`
`OF HUMAN-MILK
`L.J
`
`L_)
`
`CASEINS
`L-
`
`39
`
`MW Standard
`
`94,500
`
`55,000
`
`43,000
`
`36,000
`
`29,000
`
`18,400
`
`(cid:1) (cid:1) (cid:1) (cid:1)
`
`.
`
`,(cid:1)
`
`(cid:1)-(cid:1)----
`
`‘,
`
`i;
`
`
`(cid:1) (cid:1)..(cid:1) #{149}(cid:1) (cid:1) (cid:1)
`
`
`
`pH
`
`4.6
`
`4.3
`
`4.6
`+Ca
`
`4.3
`+Ca
`
`FIG
`I. Gel
`treated
`milk was
`6.l)containingO.
`10-20%
`polyacrylamide
`
`electrophoresis
`as indicated
`1% SDS,
`
`gel
`
`human-milk
`of
`the bottom ofthe
`at
`0.25% bromophenol
`and
`stained
`with
`
`Before
`000
`(189
`centrifugation
`casein.
`(15 (cid:1)g)diluted
`gel. Lyophilized
`casein
`blue,
`and
`2.5% /3-mercaptoethanol
`was
`Ethyl
`Stains
`All.
`
`human
`4 #{176}C;1 h) whole
`X g,
`in 0.025 mol Tris-HCI/L(pH
`subjected
`to electrophoresis
`
`in
`
`casein
`The
`(24).
`possible
`is not
`pH adjustment
`prior
`out
`contaminated
`is highly
`small
`but
`is not
`only
`very
`pellet
`to 50%,
`as could
`be shown
`by SDS-
`by whey
`proteins
`(up
`followed
`by densitometric
`determinations
`and
`PAGGE
`immunoelectrophoresis
`and
`immunodiffusion
`of mdi-
`milk
`whey
`proteins).
`Adjustment
`ofhuman
`to pH
`vidual
`4.6
`before
`ubtracentrtfugation
`to a higher
`propor-
`leads
`tion
`ofcasein
`in the
`pellet.
`improvement
`in the
`A further
`recovery
`of casein
`in the
`pellet
`is obtained
`by adjusting
`the pH to 4.3
`and,
`especially,
`by the
`addition
`of6O mmol
`Ca2(cid:1)/L
`(Fig
`1). This
`gel was
`stained
`with
`Ethyl
`Stains
`All,
`which
`has
`the
`advantage
`that
`it allows
`the
`investigator
`to
`specifically
`detect
`glycosylated
`casein
`subunits
`(K-ca-
`seins)
`and
`phosphorylated
`($-caseins)
`because
`caseins
`of
`they
`stain
`different
`colors.
`disadvantage
`A potential
`small
`this
`staining
`method
`is
`lower
`sensitivity,
`ie,
`visual-
`amounts
`ofcontaminating
`whey
`proteins
`are
`not
`ized.
`The
`effect
`ofpH
`and Ca2(cid:1)
`on whey
`contamination
`ofcasein
`pellets,
`however,
`was
`shown
`previously
`(24)
`intentionally
`overloading
`the
`gels.
`
`by
`
`a
`
`were
`
`were
`subunits
`column
`identified
`carbohydrate
`by
`caseins
`occur
`be
`expected
`dithiothreitol
`that,
`
`the
`
`the
`
`and
`was
`
`concentrations
`NaCl
`the
`used
`to
`elute
`gradient
`1 .2 mob NaCl/L
`in buffer
`
`0.6,
`(0.3,
`caseins
`B led
`
`to
`
`and
`
`of
`
`in
`
`an-
`an
`FPLC
`with
`separated
`Casein
`the
`for
`positions
`(Fig
`2). Elution
`ion-exchange
`those
`and
`analysis
`by phosphate
`/3-caseins
`in the
`fig-
`(indicated
`assay
`for
`,c-caseins
`in
`human
`in micellar
`form
`ure).
`Because
`f3-mercap-
`the
`addition
`that
`milk,
`it could
`to
`cleave
`(DTT)
`is necessary
`toethanol
`or
`would
`urea,
`combination
`with
`in
`disulfide
`bonds
`ofcasein
`subunits
`on this
`anion-ex-
`allow
`separation
`However,
`several
`experiments
`with
`change
`column.
`/3-mercaptoethanol
`or DTT
`of
`different
`concentrations
`as
`sample
`as well
`the
`run-
`to the
`to
`(0. 1% and
`1%)
`added
`the
`improve
`nor
`affect
`separation
`fling
`buffer
`did
`neither
`of
`human
`casein.
`Therefore,
`the
`subsequent
`experi-
`ments
`caseins
`were
`separated
`in
`20 mmol
`ethanol-
`amine/L
`(pH
`9.5)
`containing
`6 mob
`urea/L
`but
`without
`further
`dissociating
`agents.
`The
`effect
`of different
`1 .2 mol/L)
`in
`the
`also
`tested.
`Use
`of
`
`
`3 of 10
`
`(cid:1)
`(cid:1)
`

`
`40
`
`KUNZ
`
`AND
`
`LONNERDAL
`
`(cid:1)-
`
`q/ycosy/oted(cid:1)..-
`
`-#{248}-pPtospPicq/a/ed
`
`-(cid:1)
`
`case/n
`
`subunits
`
`Separated
`
`Separated
`
`fresh
`Ft2 days
`
`frozen
`
`-----
`
`EC
`
`0 C(cid:1)J
`
`z0 I
`
`-
`
`0(cid:1)
`a:
`
`0(
`
`I)
`
`4
`
`TIME
`
`(minutes)
`
`chromatography
`FIG 2. Anion-exchange
`a: pH 4.6;
`b: pH 4.3. Lyophilized
`casein
`added.
`Gradient
`B was used with NaCI
`
`of human-milk
`(4 mg) was
`dissolved
`from 0 to 0.6 mol/L.
`
`donor
`from one
`casein
`in 0.5 mL ethanolamine
`
`(day
`
`1 12 oflactation)
`(pH 9.5) with
`
`by FPLC.
`6 mol
`urea/L
`
`with
`mm)
`(<20
`subunits
`appro-
`the
`After
`caseins.
`were
`se-
`(cid:1)3-casein
`and
`K-
`human-milk
`same
`the
`fractions
`the whey
`with
`column
`0-1
`with
`different
`resulting
`
`(24)
`a 0-
`.2 mob
`phos-
`in
`
`casein
`ofthe
`analysis
`fast
`a very
`of
`the
`separation
`a satisfactory
`separate
`to
`priate
`conditions
`from
`fractions
`lected,
`the
`casein
`analyzed
`we
`samples
`from
`which
`anion-exchange
`the
`were
`analyzed
`on
`that,
`compared
`gradient
`0.6 mol
`NaCI/L
`in the
`elution
`ofthe
`NaCl/L,
`led to a delay
`phorylated
`and
`glycosylated
`casein
`subunits,
`enhanced
`separation
`power.
`shown
`was
`system
`FPLC
`The
`reproducibility
`ofthe
`day
`and
`same
`on the
`repeated
`injection
`sample
`ofthe
`centrifuged
`also
`We
`subsequent
`days
`not
`shown).
`(data
`the
`same
`freezing
`whole
`human
`milk
`fresh
`or after
`either
`chromato-
`The
`2).
`whole-milk
`sample
`1 12
`(Fig
`for
`by chang-
`other
`grams,
`are
`superimposed
`on each
`high
`repro-
`the
`ing
`the
`not
`only
`demonstrate
`differences
`significant
`ducibility
`that
`there
`are
`no
`also
`storage.
`after
`16 wk
`in the
`pattern
`even
`subunit
`As
`Figure
`3,
`adjustment
`of whole
`seen
`be
`man
`instead
`ofpH
`4.6
`before
`ultracentrif-
`to pH 4.3
`leads
`to more
`casein
`being
`precipitated
`but
`ugation
`to more
`whey
`proteins
`in
`the
`pellet.
`The
`presence
`whey
`proteins
`in
`the
`casein
`fractions
`was
`verified
`SDS-PAGGE
`ofthe
`anion-exchange--column
`peaks
`by
`comparing
`the
`elution
`pattern
`of
`corresponding
`whey
`fraction
`from
`the
`column
`(using
`running
`buffer
`the
`for
`casein
`separation).
`Elution
`positions
`for
`whey
`whey
`proteins
`were
`determined
`by
`running
`isolated
`pro-
`teins
`under
`these
`conditions.
`Besides
`ct-lactalbumin,
`which
`elutes
`between
`the
`glycosylated
`and
`phosphory-
`lated
`caseins,
`all major
`whey
`proteins
`show
`a shorter
`
`d
`
`which
`baseline,
`but
`
`casein
`can
`milk
`
`in
`
`the
`the
`
`by
`on
`
`hu-
`
`also
`of
`by
`and
`
`re-
`
`than
`
`the
`the
`
`ca-
`
`of cal-
`addition
`The
`show.
`caseins
`time
`tention
`insoluble
`and
`soluble
`of
`separation
`improved
`cium
`of
`increase
`a further
`was
`There
`markedly.
`proteins
`of human
`proteins
`in pellets
`ofwhey
`a decrease
`and
`seins
`(Fig
`3).
`to pH 4.3
`instead
`of4.6
`was
`adjusted
`that
`milk
`and
`casein
`superior
`separation
`of whey
`proteins
`The
`centrifuga-
`calcium
`added
`to whole
`milk
`before
`after
`was
`tion was
`used
`to isolate
`casein
`and whey
`in milk
`samples
`from one mother
`during
`the whole
`lactation
`period.
`The
`samples
`were
`subjected
`to
`FPLC
`and
`SDS-PAGGE
`as
`1,
`can
`well
`as
`to Kjeldahl
`analysis.
`As
`be
`seen
`in Table
`the
`casein
`content
`determined
`by densitometry
`varies
`be-
`22.5% and
`tween
`ofthe
`total
`protein
`content
`dur-
`45.8%
`ing
`lactation.
`The
`increasing
`concentration
`of
`casein
`during
`lactation
`and
`the
`decrease
`in whey
`proteins
`leads
`ratio
`to
`a continuous
`change
`in
`the
`of whey
`proteins
`to
`54:46
`day
`from 77:23
`on
`4 to
`on day
`35 of
`lacta-
`casein
`tion
`(Fig
`4). This
`changing
`ratio was
`confirmed
`by densi-
`tometric
`scanning
`after
`electrophoresis
`of whole
`hu-
`man
`(Table
`1).
`milk
`system
`casein
`human
`the
`The
`complexity
`of
`of whole
`casein
`SDS-PAGGE
`by
`investigated
`ther
`of
`anion-exchange-chromatogra-
`and
`different
`samples
`5). Because
`there
`are
`no commercially
`phy
`fractions
`(Fig
`the
`casein
`standards,
`the
`identity
`of
`available
`human
`to
`was
`established
`subjecting
`them
`by
`eluted
`fractions
`(Fig
`6). The
`specific
`staining
`method
`using
`SDS-PAGGE
`All
`led
`to the
`differentiation
`of glycosylated
`Ethyl
`Stains
`acid
`and
`phosphorylated
`caseins.
`Acidic
`proteins
`(sialic
`K-casein)
`stain
`blue-green
`whereas
`phosphor-
`containing
`ylated
`proteins
`stain
`blue.
`As
`an
`example,
`Figure
`5 shows
`the
`separation
`of
`casein
`sample;
`fractions
`were
`
`gel
`
`was
`
`fur-
`from
`
`a
`
`4-12
`
`
`4 of 10
`
`

`
`ANALYSIS
`
`OF HUMAN-MILK
`
`CASEINS
`b)
`
`41
`
`pH4.3-Ca
`
`pH4.3+Ca
`
`1..
`
`-
`
`E C
`
`00
`
`0
`c(cid:1)J
`
`z0 F
`
`-
`a-
`a:
`
`0 (
`
`1’)
`
`a)
`
`pH4.6-Ca
`
`pH4.61-Co
`
`1.,
`
`-
`
`E C
`
`00
`
`0
`c’J
`
`z0F
`
`-
`a-
`a:
`’)
`
`0U
`
`0.5
`
`4
`
`0.5
`
`whey
`proteIns
`
`glycosylated
`cosein
`
`phosphoryloted
`subunits
`
`10
`
`20
`
`TIME
`
`(minutes)
`
`-(cid:1)--
`
`whey
`proteins
`
`-(cid:1)---
`
`-(cid:1)
`
`glycosylated
`casein
`
`phosphorylated
`subunits
`
`10
`
`20
`
`TIME
`
`(minutes)
`
`FIG 3. Anion-exchange
`Whole
`human milk was
`gation.
`Lyophilized
`casein
`achieved
`separation
`was
`urea/L.
`
`from
`casein
`human-milk
`of
`chromatography
`addition
`adjusted
`to pH 4.6 or 4.3 with
`and without
`dissolved
`in 0.5 mL ethanolamine
`(4 mg) was
`using
`a NaC1
`gradient
`(0-0.6
`mol/L)
`in 20 mmol
`
`6 of
`(day
`donor
`one
`Ca2(cid:1)/L
`of6O mmol
`(pH 9.5) with
`6 mmol
`ethanolamine/L(pH
`
`FPLC.
`by
`lactation)
`by centrifu-
`followed
`added.
`Casein
`urea/L
`9.5)
`containing
`6 mol
`
`on SDS-
`bands
`9-
`phos-
`with
`
`or
`
`7
`
`separation
`to an electrophoretic
`subjected
`then
`blue-green
`several
`In Figure
`6 there
`are
`PAGGE.
`bands
`(fractions
`blue
`6-8)
`in
`front
`of
`the
`(fractions
`carbohydrate
`and
`is
`agreement
`with
`12), which
`before
`the
`f.(cid:1)-caseins
`phate
`analysis:
`K-caseins
`elute
`fraction.
`no cross-contamination
`in the
`6 right
`As
`can
`be
`seen
`in Figure
`SDS-PAGGE
`or K-casein
`cannot
`be used
`for quantitation
`alone
`or phosphory-
`to investigate
`changes
`in the
`glycosylation
`overlapping
`lation
`pattern
`because
`there
`are
`several
`chro-
`bands.
`We
`therefore
`FPLC
`anion-exchange
`casein
`matography
`to
`analyze
`potential
`changes
`in the
`subunit
`pattern
`occurring
`during
`lactation.
`Figure
`shows
`a chromatographic
`comparison
`between
`preterm
`with
`an
`early
`late
`casein
`sample
`from
`one
`casein
`mother.
`It
`is obvious
`there
`is an
`increase
`in casein
`content
`during
`lactation
`mainly
`because
`of
`increase
`an
`of
`the
`glycosylated
`casein
`subunits.
`The
`lack
`of K-casein
`is more
`pronounced
`in the
`premature
`casein
`sample
`than
`in the
`early milk
`sample.
`
`in
`
`lane,
`off3-
`
`used
`
`and
`that
`
`pH
`
`the
`
`contamination
`to
`4.3
`and
`an
`aggregation
`whey
`proteins
`advantages
`that
`were
`adjustment.
`ofpreparing
`of
`effectively
`chromatography,
`lower
`a significantly
`the
`chromatographic
`
`Discussion
`
`The
`human
`convenient
`
`method
`precipitation
`casein
`from whey
`to
`isolate
`
`way
`
`we developed
`proteins
`provides
`casein
`quantitatively
`
`for
`
`separating
`a rapid
`and
`without
`
`by
`
`proteins.
`by whey
`ofcalcium
`addition
`human
`of
`the
`excluded.
`are
`as compared
`based
`on
`pH
`increased
`The
`casein
`whole
`separating
`as will
`amount
`profile
`
`lower-
`The
`apparently
`subunits
`casein
`this method
`Thus,
`with
`previously
`used
`adjustment
`to
`4.6
`specificity
`the
`enhances
`pos-
`also
`subunits
`col-
`casein
`be
`described
`be-
`ofwhey
`will
`for
`
`pH
`for
`the
`by
`below,
`proteins
`individual
`
`the
`
`in
`
`significant
`ing
`ofpH
`achieves
`while
`provides
`methods
`or
`no
`method
`sibility
`umn
`cause
`obscure
`caseins.
`believed
`been
`has
`milk
`in human
`content
`casein
`The
`30% ( 19).
`on
`casein
`studies
`However,
`previous
`be
`to
`methods.
`indirect
`on
`milk
`were
`based
`in human
`content
`was
`proteins
`and whey
`In addition,
`separation
`of casein
`study
`incomplete.
`In
`this
`we
`showed
`by
`direct
`Kjeldahl
`pellets
`analysis
`of casein
`prepared
`under
`optimal
`condi-
`is marked
`tions
`there
`variation
`casein
`content
`that
`Analyzing
`during
`lactation.
`the
`casein
`pellets
`from
`one
`lactation
`of
`mother
`throughout
`shows
`22.5-458%
`the
`casein
`(Table
`total
`protein
`to be
`1). This
`is in agreement
`with
`the
`gel
`electrophoretic
`separation
`of whole
`human
`milk
`and
`determination
`relative
`distribution
`of
`in-
`ofthe
`dividual
`proteins
`densitometric
`scanning.
`This
`
`
`5 of 10
`
`(cid:17)
`

`
`42
`
`TABLE
`Protein
`
`1
`content
`
`Days of
`lactation
`
`4
`5
`6
`7
`8
`9
`10
`1 1
`12
`13
`14
`15
`16
`25
`39
`53
`67
`82
`95
`
`KUNZ
`
`AND
`
`LONNERDAL
`
`in casein
`
`pellets
`
`and whey
`
`fractions
`
`ofone
`
`mother
`
`during
`
`lactation
`
`Percent
`
`case in 0 ftotal
`
`protein
`
`Casein*
`
`gIL
`
`3.0
`3.5
`4.8
`5.6
`4.3
`4.9
`4.7
`5.4
`4.9
`5.3
`4.6
`4.9
`5.0
`5.0
`4.4
`4.0
`3.7
`3.6
`3.2
`
`Whey
`
`protein
`
`Total
`
`protein
`
`Kjeldahl
`
`Densitometer
`
`Whey-casein
`
`ratio
`
`gIL
`
`10.3
`10.2
`1 1.1
`10.0
`10.0
`9.7
`9.4
`9.3
`9.1
`9.2
`8.5
`8.9
`9.1
`8.4
`5.2
`5.4
`5.1
`4.6
`4.3
`
`gIL
`
`13.3
`13.7
`14.9
`15.6
`14.3
`14.6
`14.1
`14.7
`14.0
`14.5
`13.1
`13.8
`14.1
`13.4
`9.6
`9.4
`8.8
`8.2
`7.5
`
`%
`
`20
`20
`33
`32
`34
`28
`33
`41
`
`-
`
`38
`33
`36
`
`-
`
`40
`39
`41
`40
`44
`
`-
`
`1
`
`22.5
`25.5
`32.2
`35.9
`30.
`33.6
`33.3
`36.7
`35.0
`36.5
`35.1
`35.5
`35.5
`37.3
`45.8
`42.5
`42.0
`43.9
`42.7
`
`77:23
`75:25
`68:32
`64:36
`70:30
`66:34
`67:33
`63:37
`65:35
`63:37
`65:35
`64:36
`65:35
`63:37
`54:46
`58:42
`58:42
`56:44
`58:42
`
`milk
`
`and
`
`addition
`
`ofCaG2
`
`followed
`
`by ultracentrifugation
`
`(189
`
`000 X g,
`
`1 h,
`
`S Casein was obtained
`4 ‘C).
`
`by pH adjustment
`
`to 4.3
`
`ofwhole
`
`human
`
`content
`> 40% after
`that
`both
`principles.
`
`increases
`4 wk of
`methods
`Kjebdahl
`
`from
`lacta-
`are
`analy-
`
`50
`
`. 45
`
`. 40
`
`. 35
`
`%
`
`(cid:1)30
`
`(cid:1)25
`
`on
`
`the
`that
`shows
`4 oflactation
`day
`be
`emphasized
`should
`It
`on completely
`different
`
`casein
`to
`
`method
`(cid:1)20%
`tion.
`based
`
`E E
`
`6
`
`5.
`
`4.
`
`3.
`
`2
`
`1
`
`0
`
`0
`
`,
`
`(cid:1)t
`
`20
`
`a conver-
`requires
`content,
`nitrogen
`the
`sis, by measuring
`The most
`content.
`protein
`the
`calculate
`sion
`factor
`to
`are
`6.38
`or
`proteins
`for milk
`factors
`commonly
`used
`study
`as well;
`in this
`was
`used
`factor
`and
`the
`batter
`6.25,
`for
`caseins,
`is
`in particular
`factor,
`however,
`the
`correct
`into
`account
`the
`complex
`composi-
`not
`known.
`Taking
`the
`true
`conversion
`factor
`is likely
`tion
`ofhuman
`casein,
`to be different
`from those
`currently
`used;
`however,
`this
`factor
`cannot
`be determined
`yet because
`exact
`carbo-
`hydrate
`composition
`of
`subunits
`their
`variable
`the
`with
`amino
`sugar
`contents
`unknown.
`Glycoproteins
`may
`are
`also
`stain
`slightly
`differently
`(depending
`on degree
`of gly-
`cosylation)
`after
`gel
`electrophoresis
`and might
`therefore
`have
`an
`influence
`on
`densitometric
`scanning.
`Despite
`the
`different
`techniques
`used,
`results
`both
`methods
`are
`in good
`agreement.
`lactation
`during
`The
`increase
`in casein
`content
`by the maturation
`nounced
`and
`is likely
`to be caused
`exact mechanisms
`the mammary
`gland;
`however,
`the
`not
`known.
`is obvious
`that
`the
`synthesis
`ofcaseins
`whey
`proteins
`is regulated
`by different
`mechanisms
`most whey
`proteins
`decrease
`in concentration
`cause
`ing
`lactation
`(33).
`The
`decrease
`of whey
`proteins
`leads
`1) and
`the
`increase
`in
`casein
`during
`lactation
`The
`ratio
`continuously
`changing
`whey-to-casein
`ratio.
`(cid:1)-‘-‘60:40
`that we
`found
`in our
`study
`for mature
`human
`the
`milk
`is the
`same
`as
`ratio
`that,
`despite
`the
`back
`of a
`direct
`method
`to determine
`the
`amount
`ofcasein
`in hu-
`man milk,
`been
`recommended
`as a guideline
`for
`the
`has
`preparation
`ofinfant
`formulas.
`
`the
`
`our
`
`from
`
`is pro-
`of
`are
`and
`be-
`dur-
`(Table
`to
`
`a
`of
`
`It
`
`___________________
`
`I
`
`40
`
`#{149}
`
`I
`60
`
`#{149}
`
`U
`
`80
`
`(cid:1) (cid:1) 20
`100
`
`day of lactation
`
`FIG 4. Casein
`(%) of
`total milk
`tion.
`
`(0 -
`concentration
`its
`and
`0)
`(#{149}
`#{149})
`protein
`for one mother
`
`-
`
`proportion
`relative
`throughout
`lacta-
`
`
`6 of 10
`
`

`
`ANALYSIS
`
`OF HUMAN-MILK
`
`CASEINS
`
`43
`
`(cid:1)-
`
`glycosylated
`
`-(cid:1)
`
`*-
`
`phosphorylated
`
`-(cid:1)
`
`casein
`
`subunits
`
`371
`
`2..
`
`1.2
`
`E C
`
`0C
`
`O
`csJ
`
`z0 I
`
`-
`a-
`a:
`
`0U
`
`)
`CO
`4
`
`FIG 5. Anion-exchange
`conditions
`were
`the
`
`same
`
`chromatography
`as in Figure
`
`2.
`
`ofa
`
`human-milk
`
`casein
`
`sample(day
`
`12 oflactation)by
`
`FPLC.
`
`Separation
`
`TIME
`
`(minutes)
`
`MW
`
`Standard
`
`95,500
`
`55,000
`
`43)000
`
`36,000
`
`29,000
`
`18,400
`
`12,400
`
`.$(cid:1)-
`
`::::
`
`I
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`electrophoresis
`FIG 6. Gel
`buffer was added
`sample
`see Fig
`staining
`1.) Left
`
`offractions
`to 40 ML sample,
`lane, molecular-weight
`
`from Figure
`4-12
`and a l5-(cid:1)iL portion
`markers;
`right
`
`eluted
`5. The
`was loaded
`lane, whole
`
`concentrated,
`were
`fractions
`FPLC
`separation
`conditions
`on the gel.
`(For
`before
`the FPLC separation.
`casein
`
`60 (cid:1)zL
`and
`
`
`7 of 10
`
`(cid:1)
`

`
`44
`
`KUNZ
`
`AND
`
`LONNERDAL
`
`..-
`
`g/ycosy/oted(cid:1)-
`
`-(cid:1)pbusphory/oted-#{248}
`
`casein
`
`subunits
`
`-
`
`---
`
`-.-.
`
`Premature
`mi/k
`/octoti#{244}n
`1st day of
`i(cid:1)ern
`day of/ac/a/ion
`
`E C
`
`0a
`
`,
`CsJ
`
`2.(cid:1)
`
`z0 I
`
`-a
`
`-
`a:
`
`0U
`
`)
`CO
`4
`
`TIME
`
`(minutes)
`
`FIG 7. Anion-exchange
`premature
`milk
`(one
`same
`as in Figure
`2.
`
`chromatography
`and
`early
`
`and
`
`human-milk
`of
`late milk
`samples
`
`FPLC.
`by
`casein
`from another
`
`Casein
`mother.
`
`samples
`Separation
`
`prepared
`were
`conditions
`
`from
`the
`
`were
`
`donor)
`
`an
`using
`whey
`
`to
`
`FPLC
`that
`demonstrated
`previously
`(24)
`We
`human
`separate
`efficiently
`can
`exchanger
`anion
`anion-exchange
`same
`the
`we used
`study
`In this
`proteins.
`buffer
`was
`needed
`A urea
`separation.
`for
`casein
`column
`spontaneously
`otherwise
`that
`subunits
`to disassociate
`the
`was
`used
`or micelles.
`FPLC
`to submicelles
`aggregate
`chromato-
`subunits
`(34-37).
`The
`separate
`bovine
`casein
`in
`general
`human
`casein
`is
`of
`graphic
`separation
`(38-42).
`cellulose
`chromatography
`achieved
`by DEAE
`and
`are
`very
`time
`consuming
`separations
`less
`Those
`and
`to peak
`resolution.
`Davies
`powerful
`with
`regard
`Law
`Q
`(37),
`comparing
`DEAE-cellubose
`with
`FPLC-Mono
`chromatography
`ofbovine
`casein
`mixtures,
`found
`a high
`correlation
`coefficient
`between
`both methods.
`The major
`difference
`from
`our
`FPLC
`method
`(besides
`a different
`buffer
`composition
`and
`slightly
`different
`running
`condi-
`tions)
`is their
`need
`to alkylate
`whole
`bovine
`casein,
`which
`beads
`to an
`improved
`resolution
`the
`different
`peaks.
`This might
`be due
`to a more
`complex
`casein
`composition
`and
`‘y-casein)
`of
`com-
`/3-,
`bovine
`(asi-,
`casein
`aS2,
`K-,
`pared
`with
`human
`casein
`((cid:1)3-
`and
`Dalgleish
`K-casein).
`(36)
`reported
`the
`ofFPLC
`with
`the Mono-Q
`(anion-
`use
`exchange)
`and
`Mono-S
`(cation-exchange)
`columns
`mainly
`to study
`some
`characteristics
`ofglycosylated
`and
`K-casein
`from
`bovine
`milk.
`The
`basic
`unglycosylated
`difference
`from our method
`is that
`addition
`of a re-
`ducing
`agent
`(eg,
`fl-mercaptoethanol)
`is necessary
`for
`separation
`ofthe
`casein
`subunits.
`is interesting
`to note
`It
`use
`that
`in our
`study
`we
`found
`the
`ofreducing
`agents
`((cid:1)3-
`mercaptoethanol
`and
`DTT)
`unnecessary
`the
`separa-
`human
`tion
`of
`the
`casein
`subunits.
`agents
`were
`used
`commonly
`for bovine
`casein
`and
`it was
`
`of
`
`the
`
`for
`
`These
`separation,
`
`used
`
`plate.
`when
`all proteins,
`
`in this
`to directly
`phosphorylated
`and
`therefore
`We
`comparing
`not
`
`expect
`large
`the
`
`only
`
`characteristic
`are
`groups
`sulthydryl
`free
`that
`shown
`may
`contribute
`therefore
`subunits
`and
`casein
`some
`groups
`may
`sulfhydryb
`formation.
`Thus
`micellar
`formation
`for
`human
`casein
`micelle
`significant
`is the
`micelles.
`Our
`reported
`method
`bovine
`both
`that
`in one
`rapid
`step
`separates
`method
`which
`of human
`milk,
`the
`(cid:1)3-caseins
`and
`seins
`in
`glycosylation
`and
`phosphorylation
`complex
`milk.
`Previously,
`/3-caseins
`were
`than
`bovine
`arately
`(1 , 23)
`and
`the
`separation
`ofK-caseins
`to be very
`difficult
`(2 1, 22,
`43).
`42,
`sub-
`casein
`in
`There
`are
`pronounced
`differences
`and mature
`unit
`pattern
`for premature
`milk,
`cobostrum,
`are present
`milk.
`In premature
`milk
`the K-casein
`subunits
`They
`are
`also
`in
`very
`low concentrations
`or
`are missing.
`lower
`in colostrum
`although
`a subunit
`pattern
`more
`sim-
`This
`ilar
`to that
`of mature
`milk
`is established.
`suggests
`hormonal
`control
`of
`the
`synthesis
`of K-casein.
`The
`sub-
`unit
`pattern
`for
`to be established
`in pre-
`/3-casein
`appears
`mature
`milk,
`but
`the
`concentrations
`ofmost
`subunits
`are
`higher
`in cobostrum
`and
`in mature
`milk.
`This
`also
`sug-
`gests
`hormonal
`regulation
`offl-casein
`synthesis.
`Whether
`and
`both
`protein
`synthesis
`side-chain
`modification
`(gly-
`are
`cosylation
`and
`phosphorylation)
`of
`the
`subunits
`in-
`fluenced
`by the
`regulatory
`mechanisms
`needs
`to be stud-
`ied further.
`stain
`The
`specific
`opportunity
`provides
`an
`lic acid-containing)
`in the
`same
`gel
`technique
`useful
`pbes. Because
`
`of
`to
`less
`be
`for
`than
`first
`FPLC
`the K-ca-
`are more
`pattern
`studied
`sep-
`has
`proven
`
`the
`
`All,
`Stains
`(sia-
`acidic
`subunits
`become
`of sam-
`and
`
`a
`
`Ethyl
`study,
`compare
`casein
`it
`to
`numbers
`glycosybated
`
`
`8 of 10
`
`

`
`ANALYSIS
`
`OF HUMAN-MILK
`
`CASEINS
`
`45
`
`ad-
`
`be-
`it
`is
`
`this
`
`detected,
`Coomassie
`are
`several
`f3-casein
`reported
`by
`gel
`scanning
`eg,
`by
`
`provides
`stain
`It
`is obvious
`Blue.
`bands
`K-casein
`band.
`Therefore
`(44-46),
`to
`quantitate
`electrophoresis
`of whole
`without
`further
`sepa-
`chromatographic
`tech-
`
`are
`ones,
`phosphorylated
`like
`stains
`over
`vantages
`there
`6 that
`Figure
`from
`prominent
`the most
`hind
`although
`advisable,
`not
`f3-casein
`or K-casein
`only
`with
`following
`gel
`casein
`ration
`of
`the
`subunits,
`niques.
`separat-
`of
`the methods
`that
`we believe
`In conclusion,
`the
`casein
`quantitatively,
`separating
`ing whole
`human
`chroma-
`and
`/3-casein
`subunits
`by FPLC
`anion-exchange
`PAGGE
`in
`tography
`and
`detecting
`the
`same
`subunits
`to
`capacity
`gels,
`provide
`significant
`improvements
`in our
`and
`caseins
`study
`the
`structure
`function
`human
`of
`of
`purpose
`the
`variations
`during
`lactation.
`The major
`We
`this work
`was
`to develop
`the
`separation
`techniques.
`applied
`these
`techniques
`to
`several
`samples
`obtained
`from one
`donor
`throughout
`lactation
`and
`to samples
`tamed
`from mothers
`delivering
`prematurely.
`
`and
`
`K-
`
`ob-
`Cl
`
`amino
`casein:
`Eur
`J Biochem
`D,
`Lifchitz
`properties
`from human
`
`sequence,
`acid
`145:677-82.
`1984;
`A, Deleur#{233}J, Floc’h
`and
`three-dimensional
`and
`cow caseins.
`
`synthesis
`
`F,
`
`FEBS
`
`in
`
`normal
`
`and
`
`abnormal
`
`and
`J Dairy
`
`albu-
`serum
`Sci
`l974;57:
`
`K. Molecular
`its interaction
`l984;48:77l-6.
`ofhuman
`
`weight
`with
`other
`
`and
`hu-
`
`milk. Am J Clin
`
`B. Quantification
`G, Ribadeau-Dumas
`J Dairy Res
`l985;52:239-47.
`S.
`H, Kaminogawa
`N, Kobayashi
`J Biochem
`198 l;90:l005-12.
`casein.
`A, Ribadeau-Dumas
`B. Preparation
`ofhuman
`Lett
`l985;l88:48-
`sc-caseifl.
`
`of
`
`fi-
`
`Isolation
`
`and
`
`FEBS
`
`from human
`hexapeptide
`properties.
`and
`biological
`Berthou
`J, Migliore-Samour
`Joll#{233}sP.
`Immunostimulating
`structures
`of
`two
`tripeptides
`Lett
`1987;2l8:55-8.
`distribution
`protein
`The
`Rowland
`SJ.
`l938;9:47-57.
`Res
`milk.
`J Dairy
`J, Takase M. Lactoferrin
`1 7. Nagasawa
`T, Kiyosawa
`mm ofhuman
`casein
`in colostrum
`and milk.
`I 159-63.
`5, Yamauchi
`1 8. Azuma
`N, Kaminogawa
`and
`conformation
`ofhuman
`,c-casein
`man milk
`proteins.
`Agric Biol Chem
`19. L#{246}nnerdalB, Forsum
`E. Casein
`content
`Nutr 1985;41:l 13-20.
`Chtourou
`A, Brignon
`casein
`human
`milk.
`Yamauchi
`K, Azuma
`and
`propertiesofhuman
`22. Brignon
`G, Chtourou
`amino
`acid
`sequence
`54.
`Greenberg
`identification
`
`15.
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`20.
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`21.
`
`23.
`
`in
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`References
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`
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`
`C,
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`l984;259:
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`
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