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`
`THE DENATURATION
`
`OF PROTEINS
`REVERSAL*
`
`AND
`
`ITS APPARENT
`
`II. HORSE
`
`SERUM
`
`PSEUDOGLOBULIN
`
`BY HANS
`
`NEURATH,
`
`GERALD
`
`(From
`
`the Department
`
`of Biochemistry,
`Durham,
`
`0. ERICKSON
`AND JOHN
`R. COOPER,
`Duke University School
`of Medicine,
`Carolina)
`
`North
`
`(Received
`
`for
`
`publication,
`
`June
`
`9, 1941)
`
`In the preceding paper (2) the denaturation of horse serum al-
`bumin by urea and guanidine hydrochloride and its apparent re-
`versal were described. These studies have been extended in the
`present investigation
`to pseudoglobulin components with
`the
`object of determining any differences that may exist in the de-
`naturation process of the protein constituents of normal horse
`serum. Quantitative studies have been made possible by recent
`improvements
`in the experimental methods of isolating mono-
`disperse serum pseudoglobulin fractions
`(3, 4). Their known
`physical and chemical characteristics serve as a sensitive criterion
`for the extent to which denaturation can be reversed.
`
`EXPERIMENTAL
`
`Material
`The method of preparation of monodisperse fractions of serum
`pseudoglobulins by means of fractional precipitation with am-
`monium sulfate under defined experimental conditions relative
`to protein concentration, pH, and salt concentration has been
`described in a previous publication
`(3). The present measure-
`ments were carried out by the technique already described (2) with
`the pseudoglobulins GI and GII, precipitable by ammonium sul-
`fate at pH 6.4 within
`the limits of 1.1 to 1.36 and 1.4 to 1.6 M
`respectively. The molecular weights of GI and GII were found
`
`* Presented
`of Biological
`
`the Thirty-fifth
`at
`Chemists
`at Chicago,
`
`meeting
`15-19,
`
`of
`1941
`
`the American
`(1).
`
`Society
`
`annual
`April
`265
`
`KASHIV EXHIBIT 1061
`IPR2019-00791
`
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`
` by guest on September 27, 2018
`
`266
`
`Denaturation
`
`of Proteins.
`
`II
`
`in satis-
`to be 170,000,
`and viscosity measurements
`by diffusion
`factory
`agreement with
`the values of 165,000 obtained by Tiselius
`for electrophoretically
`isolated material
`(5), and of 178,000 found
`by Burk
`(6) from osmotic pressure measurements.’
`fundamental
`Since
`it was
`found early
`in
`this work
`that no
`differences existed between
`the fractions GI and GII
`relative
`to
`the denaturation
`process as studied here, most of the measurements
`described below were carried out with
`the pseudoglobulin GII, as it
`was available
`in larger quantities.
`
`Results
`in Presence of Urea and Guanidine
`Denatured Pseudoglobulin
`Hydrochloride-The
`diffusion and viscosity
`of pseudoglobulin were
`measured
`in the presence of different amounts of urea and guani-
`dine hydrochloride
`in solutions
`containing
`0.05 N acetate buffer
`and 0.2 M NaCl at pH 5.5. The results of the viscosity determina-
`tions carried out at protein concentrations
`between 0.1 and 1.2 per
`cent are plotted
`in Fig. 1.
`the relative
`(2),
`As in the analogous
`studies on serum albumin
`of the denaturing
`viscosities
`increase with
`increasing
`concentration
`agent,
`the increase being greater
`for guanidine hydrochloride
`than
`for comparable
`concentrations
`of urea.
`The limiting slopes of the
`curves were determined
`from
`the intercept when qsP/c was plotted
`against c, where qsP is the specific viscosity
`and c the protein con-
`centration
`in weight per cent (7). Comparative measurements
`in
`the pressure
`viscometer
`showed
`the relative
`viscosities
`to be in-
`dependent
`of the velocity
`gradient within
`the region of 175 to
`2000 sec.-‘.
`the
`with
`in conjunction
`were measured
`constants
`Diffusion
`in Table
`I.
`The results are summarized
`viscosity
`determinations.
`curves
`indicated
`the solutions
`to be
`Analysis
`of the diffusion
`essentially monodisperse
`in urea concentrations
`of 5 and 8 M, and
`in guanidine hydrochloride
`concentrations
`of 2 and 5.6 M.
`In
`0.5 and 3 M guanidine hydrochloride
`there is some spreading of the
`values as determined
`by
`the method of successive
`analysis,
`in-
`dicating
`the presence of a small fraction of lower diffusion constant.
`Calculations
`of apparent molecular shapes and molecular weights
`1 Lower values, i.e. 142,000,
`have been reported by Cohn et al. (4)
`for
`material isolated by methods similar to those employed by the authors (3).
`
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`
`Neurath, Cooper, and Erickson
`
`267
`
`in
`data were carried out as described
`from diffusion and viscosity
`the preceding paper
`(2) and are summarized
`in Table
`II’.
`There
`are also included values
`for the molecular
`shape,
`(b/~)~, calculated
`with
`the assumption
`of 33 per cent hydration.
`The empirical
`
`l.36-.
`
`1.28-
`
`1.24-.
`
`1.20--
`
`I .os--
`
`;
`z
`0
`::
`5
`
`GUANIDINE
`
`HCL
`
`0:2
`
`0:a
`PERCENT
`
`016
`
`6.6
`PROTEIN
`
`1.0
`
`by urea and
`denatured
`viscosities of pseudoglobulin
`JGo. 1. Relative
`concentration
`in weight
`guanidine
`hydrochloride,
`plotted
`against protein
`per cent. Open circles refer to 3 M guanidine
`hydrochloride,
`open squares
`to 8 M urea, open
`triangles
`to 5.6 M guanidine
`hydrochloride,
`solid circles
`to 0.5 M guanidine
`hydrochloride,
`solid squares
`to 5 M urea, and solid
`triangles
`to 2 M guanidine
`hydrochloride.
`
`viscosity correction for the diffusion constant (2) was applied to
`the measurements in urea solutions and to the measurements in
`guanidine hydrochloride
`in concentrations higher than 2 M.
`In
`cases in which the solutions were found to be polydisperse, the
`limiting value of the diffusion constant was used for molecular
`weight calculations.
`
`Page 3
`
`

`

`of Denatured
`Denatured
`D = mean
`in seconds;
`time
`corrected
`diffusion
`constant
`the
`1); D,, D,,
`and Da are
`height,
`standard
`deviation,
`Unless
`otherwise
`indicated,
`values
`determined
`from
`evenly
`
`Diffusion
`
`Constants
`
`t =
`=
`D’
`Equation
`maximum
`respectively.
`about
`six
`curves
`(3).
`
`I
`TABLE
`“Reversibly”*
`and
`Pseudoglobulin
`second;
`in sq. cm. per
`diffusion
`constant
`of
`the
`solvent
`((2)
`the
`for
`viscosity
`calculated
`the
`by
`diffusion
`constants
`analysis
`methods,
`and
`successive
`each
`D3 value
`is
`the mean
`spaced
`parts
`of
`the
`diffusion
`
`and
`
`Irreversibly
`
`of
`
`Downloaded from
`
`http://www.jbc.org/
`
`,ry%Y--n~
`
`t
`
`/ D1
`
`/ Dz 1
`
`D,
`
`Pseudoglobulin
`
`in 0.5 M gusnidine HCl
`
`Pseudoglobulin
`
`Per
`cent
`0.5
`
`sec.
`23,640
`34,980
`82,140
`
`10-V
`
`4.16
`4.02
`3.91
`
`lo-’
`
`10-1
`
`4.02
`
`3.934.4:
`
`Limiting
`value.....
`D’..........
`
`IO-’
`4.4 X
`4.6 X 1OP
`
`Pseudoglobulin
`
`in 2 M guanidine HCl
`
`in 5 M urea
`-_____--_
`10-7
`
`lo-’
`
`2.72
`
`Per cent
`
`1.2
`
`sec.
`
`38,640
`75,000
`104,400
`164,100
`
`10-7
`
`2.97
`2.75
`2.77
`2.65
`
`Average.
`D’.
`
`2.76 X
`3.73
`X
`
`lo-’
`lo-’
`
`f
`
`0.12
`
`Pseudoglobulin
`
`in 8 M ures,
`
`0.55
`
`24,720
`32,640
`41,580
`73,380
`
`2.76
`2.86
`2.87
`2.79
`
`1.2
`
`2.89
`
`1.79
`
`1.89
`
` by guest on September 27, 2018
`
`86,400
`109,440
`132,420
`51,165
`79,380
`103,680
`
`1.76
`1.68
`1.75
`1.65
`1.73
`1.62
`
`Average.
`D’
`
`1.70
`2.97
`
`x
`x
`
`lo-’
`10-T
`
`zk 0.09
`
`Pseudoglobulin,
`
`“reversibly”
`by 8 M urea
`
`denatured
`
`35,760
`45,600
`59,940
`71,940
`
`4.56
`4.44
`4.49
`4.61
`
`4.50
`
`4.55
`
`Average.
`D’.
`
`4.53
`4.69
`
`x
`X
`
`lo-’
`lo-’
`
`f
`
`0.12
`
`denatured
`irreversibly
`Pseudoglobulin,
`by 7.5 iv urea, at pH 7.5
`
`Limiting
`value.....
`
`D’
`
`3.0
`3.7
`
`x
`x
`
`10-T
`10-1
`
`Pseudoglobulin
`
`in 5.0 M guanidine HCl
`
`Average..
`D’.
`
`2.50
`4.02
`
`X 10-r
`X
`lo-’
`
`rfr 0.11
`
`* See
`
`(2),
`
`foot-note
`
`2.
`
`268
`
`Average.
`D’.
`
`2.85
`3.19
`
`X
`X
`
`lo-’
`lo-’
`
`f
`
`0.05
`
`-
`
`Pseudoglobulin
`
`in 3 M guanidine HCl
`
`0.8
`
`20,520
`20,400
`27,000
`64,200
`81,120
`
`2.88
`2.67
`2.72
`2.63
`2.75
`
`2.5833.01
`2.61-3.0:
`
`0.7
`
`Page 4
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`
`Neurath, Cooper, and Erickson
`
`269
`
`Denatured
`Irreversibly
`and
`between L’Reversibly”
`Distribution
`Pseudoglobulins-Removal
`of urea or guanidine hydrochloride
`by
`dialysis against distilled water
`in the cold resulted
`in partial pre-
`cipitation
`of the proteins.
`The precipitate was of gelatinous
`con-
`sistency
`varying
`in amount with
`the pH of the suspension and with
`
`Molecular
`
`Constants
`
`and
`
`“Reversibly”
`
`Denatured
`
`II
`TABLE
`.Denatu,red,
`oj” Native,
`Pseudoglobulins
`viscosity
`the specific
`when
`slope of the curves
`obtained
`limiting
`the
`qap/c =
`axes
`for
`the
`ratio
`of
`the
`against
`protein
`concentration;
`b/a
`=
`is plotted
`solvation
`ellipsoid,
`calculated
`with
`the Simha
`viscosity
`equation,
`prolate
`neglected;
`(b/a),
`=
`the axial
`ratio
`calculated
`for 33 per cent hydration;
`being
`the
`diffusion
`constant
`corrected
`for
`the
`viscosity
`of
`the
`solvent
`=
`D’
`Equation
`1);
`(J/fo)
`=
`l,he
`dissymmetry
`constant
`calculated
`from
`((2)
`viscosity
`data;
`and M =
`the molecular
`weight
`calculated
`from
`diffusion
`and
`viscosity
`data.
`
`a
`
`Protein
`
`HCl..
`HCl
`I‘
`
`HCl
`
`Native.....................
`In 0.5 M guanidine
`“
`2 M guanidine
`“
`3 “
`“
`5.6 M guanidine
`“
`“ 5 M urea..
`‘I 8 “
`“
`“Reversibly”
`8 ivr urea
`
`6.60
`7.75
`14.90
`27.0
`27.0
`14.0
`.
`.
`. . . . . . . . 28.0
`. . .
`. . . .
`denatured
`by
`..___.._.
`,__._
`
`5.90
`
`7.2
`8.1
`13.0
`19.0
`19.0
`12.4
`19.5
`
`5.2
`6.0
`10.0
`15.0
`15.0
`9.3
`15.4
`
`10-7
`
`4.75
`4.6*
`3.19
`3.7*
`4.02
`3.73
`2.97
`
`1.39
`1.44
`1.69
`1.95
`1.95
`1.66
`1.98
`
`170,000
`170,000*
`307,000
`95,9OO*t
`74,800j
`152,ooot
`170,000t
`
`6.6
`
`4.7
`
`4.69
`
`1.35
`
`190,000
`
`solution
`* The
`the
`limiting
`refer
`to
`analysis.
`cessive
`t Molecular
`diffusion
`constant
`
`somewhat
`was
`diffusion
`
`The
`I).
`(see Table
`polydisperse
`constant
`determined
`by
`the method
`
`values
`of suc-
`
`weight
`(see
`
`calculated
`the
`text).
`
`with
`
`the
`
`empirical
`
`correction
`
`for
`
`the
`
`yield was obtained when solutions were
`Maximum
`temperature.
`adjusted
`to pH 6.0 and stored at about 0”.
`(irreversibly
`The quantitative
`distribution
`between
`insoluble
`denatured)
`and soluble
`(“reversibly”
`denatured)
`pseudoglobulin
`was studied as a function
`of the concentration
`of the denaturing
`agent originally
`present.
`10 cc. samples of 2 per cent protein
`in
`2, 4, 6, 7, and 8 M urea or guanidine hydrochloride
`were prepared
`
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` by guest on September 27, 2018
`
`270
`
`Denaturation
`
`of Proteins.
`
`II
`
`in the ice box against running distilled
`and, after 12 hours, dialyzed
`The solutions were next adjusted
`to
`water until
`free from salt.
`at 0”.
`The precipitates
`were
`then
`pH 6.0 and stored overnight
`centrifuged,
`washed
`once with
`distilled water,
`dissolved
`in a
`phosphate
`buffer of pH 7.1, and made
`to volume.
`The super-
`natants and washings were
`likewise made to volume and the pro-
`
`GUANIDINE
`
`/-
`
`9
`I) 8~
`t
`5 0 lo-
`3
`m
`2 60-
`w
`2
`$ so-
`z
`p 2 40-
`
`HCL I PSEUDOGLOBULIN
`
`,
`
`,
`,
`IO 12
`
`HCL
`
`q//J//
`
`2
`MOLARITY
`
`4
`OF UREA
`
`6
`
`8
`OR GUANIDINE
`
`FIG. 2. The
`fraction
`against
`dialysis,
`plotted
`at which
`denaturation
`to guanidine
`hydrochloride,
`
`of
`
`pseudoglobulin
`total
`the molarity
`of urea
`occurred.
`circles
`
`Open
`
`irreversibly
`or guanidine
`refer
`to urea,
`
`denatured
`hydrochloride
`solid
`circles
`
`after
`
`tein concentrations in the respective fractions determined with the
`Koch-McMeekin method (8). The results of these measurements,
`carried out in triplicate, are given in Fig. 2 in which the fraction of
`total protein irreversibly denatured is plotted against the concen-
`tration of urea, or guanidine hydrochloride originally present.
`“Reversibly” Denatured Pseudoglobulin--In
`the following ex-
`periments, a 2 per cent protein solution was denatured by 8 M urea
`
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`
`Neurath, Cooper, and Erickson
`
`271
`
`removed by dialysis.
`for 24 hours, urea was
`and, after standing
`Diffusion measurements
`on the supernatant
`solution, obtained after
`precipitation
`of the irreversibly
`denatured protein at pH 6.0, indi-
`cated
`the material
`to be polydisperse.
`The diffusion
`constants,
`measured
`in the presence of an acetate buffer of pH 5.5, varied be-
`tween 3.8 X lop7 near the peak of the diffusion
`curves and 4.6 X
`lo-’
`in the
`lower
`regions.
`The
`limiting
`slope of the viscosity
`curves, qsp/c, was
`found
`to be higher
`than that of the native globu-
`lin; i.e., 7.90 as compared with 6.60.
`in the super-
`contained
`For
`further
`purification,
`the proteins
`natant
`solution were
`subjected
`to
`fractional
`precipitation
`with
`for
`ammonium
`sulfate,
`according
`to
`the method employed
`the
`purification
`of the native protein:
`the protein
`concentration
`was
`adjusted
`to 3 per cent, the pH
`to 6.4, and the ammonium
`sulfate
`At
`this point
`the
`concentration
`gradually
`increased
`to 1.1 M.
`solution
`became slightly
`opalescent
`and when more ammonium
`sulfate was added, up to 1.36 M, a precipitate
`settled out.
`After
`the precipi-
`filtration,
`the salt concentration
`was
`raised
`to 1.6 M;
`tate collected by centrifugation,
`dialyzed, and, after
`removal of
`traces of euglobulin by pH adjustment
`to 6.2 and 5.0, used
`for
`diffusion
`and viscosity measurements
`in the presence of a 0.05
`M acetate buffer, pH 5.5, containing 0.2 M NaCl.
`in Table I.
`The results of the diffusion measurements
`are listed
`The material proved
`to be monodisperse with a diffusion
`constant
`of D = 4.69 X 1O-7 with a standard
`deviation
`of the mean of
`SO.12
`x 10-7.
`The
`limiting
`slope of the viscosity
`curves,
`il-
`lustrated
`in Fig. 3, was 5.90 as compared with 6.60
`for native
`material.
`The molecular weight of 190,000 calculated
`from
`these
`data
`is somewhat
`higher
`than
`that
`found
`for
`the native material
`(Table
`I).
`fraction which pre-
`Irreversibly Denatured Pseudoglobulins-The
`cipitated upon adjustment
`of the pH
`to 6.0, following
`removal of
`8 M urea by dialysis, was considered
`to be irreversibly
`denatured
`material.
`For
`further purification,
`it was dissolved by acidifying
`In a
`to pH 4.0 and freed from any insoluble
`residue by filtration.
`concentration
`of 2 per cent protein,
`the solution was highly viscous
`It did not show
`and appeared
`to exhibit
`thixotropic
`properties.
`double refraction
`of flow
`in the apparatus
`described by Edsall and
`Mehl
`(9).
`The protein was
`reprecipitated
`by adjustment
`of the
`
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`
` by guest on September 27, 2018
`
`272
`
`Denaturation
`
`of Proteins.
`
`II
`
`Addition of the
`at the desired pH.
`to 6.0 and then redissolved
`pH
`precipitation.
`resulted
`in partial
`buffer components
`by dialysis
`The results of the viscosity measurements
`carried out
`in a 0.05
`N acetate buffer at pH 4.12, containing 0.1 N NaCl, and in a 0.02
`N veronal-acetate
`buffer at pH 7.5, containing
`0.1 N NaCI, are
`shown
`in Fig. 3. The relative
`viscosities were also measured
`in
`t,he pressure viscometers
`and found
`to be independent of the veloc-
`
`1.24
`
`?
`,
`
`--
`
`1.20
`
`!
`
`>
`c
`in
`0
`
`DENATURED
`
`0
`
`0.2
`
`0.6
`0.4
`PERCENT
`
`0.8
`PROTEIN
`
`1.0
`
`1.2
`
`of
`
`viscosities
`3. Relative
`FIG.
`plotted
`against
`pseudoglobulin,
`refer
`irreversibly
`cent.
`Triangles
`to
`to
`the
`same
`protein
`at pH
`7.5,
`circles
`purified
`by
`fractional
`precipitation
`with
`For
`comparison,
`the
`slope
`of
`the
`viscosity
`also
`indicated.
`
`of
`
`“reversibly”
`and
`irreversibly
`concentration
`the
`protein
`protein
`denatured
`“reversibly”
`to
`ammonium
`curve
`
`denatured
`per
`in weight
`at pH 4.1, squares
`denatured
`protein,
`sulfate
`(see
`the
`text).
`the
`native
`protein
`
`is
`
`The rate of diffusion,
`ity gradient? between 400 and 2500 sec.-l.
`At pH 4.12 no measurable
`like the viscosity, was a function of pH.
`rate could be observed after 72 hours diffusion of a 0.3 per cent
`At pH 7.5, the calculated mean
`solution,
`indicating gel formation.
`diffusion
`constant was
`considerably
`lower
`than
`that of the de-
`
`is
`2 There
`tected
`at very
`Couette
`apparatus
`
`little
`low
`
`doubt,
`velocity
`are under
`
`that
`
`be de-
`may
`viscosity
`structural
`however,
`in a modified
`Such measurements
`gradients.
`way
`and will
`be reported
`elsewhere.
`
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`Neurath, Cooper, and Erickson
`
`273
`
`varied
`The calculated D3 values
`in 8 M urea.
`natured protein
`widely
`from each other, probably due to restriction
`of free diffusion
`(Table
`I).
`
`DISCUSSION
`
`to
`is analogous
`effect of urea on pseudoglobulin
`The denaturing
`for
`serum
`albumin.
`The apparent molecular
`that
`observed
`asymmetry
`increases with
`increasing
`concentration
`of urea, while
`the molecular weight
`remains essentially
`unchanged.
`This
`latter
`finding
`is in agreement with
`the osmotic pressure measurements
`of
`Burk
`(6).
`The solutions
`of the denatured
`protein
`in urea are
`monodisperse,
`indicative of a uniform action of urea on all pseudo-
`globulin molecules.
`are more com-
`The effects produced by guanidine hydrochloride
`guanidine
`produces
`In 0.5 M concentration,
`only minor
`plex.
`changes
`in apparent molecular
`shape and no changes
`in molecular
`weight.
`Diffusion measurements
`indicate, however,
`the presence
`In 2 M solution,
`the ap-
`of material of higher molecular weight,
`parent molecular asymmetry
`of the protein
`is markedly
`increased
`and the molecular weight
`is nearly
`twice
`that of the native protein.
`When
`the guanidine hydrochloride
`concentration
`is increased
`to
`the molecular asymmetry
`becomes drastically
`increased and
`3 M,
`about equal to that produced by 8 M urea solutions.
`The diffusion
`constant,
`however,
`does not decrease
`in proportion
`but, on the
`contrary,
`increases.
`The mean molecular weight,
`calculated
`from
`values of ~~~/c and D is about one-half of that of the
`the limiting
`native protein.
`Further
`increase
`in guanidine hydrochloride
`con-
`to 5.6 M, produces
`no further
`changes
`in apparent
`centration,
`molecular shape or molecular weight except that
`the solutions now
`become monodisperse
`(Table
`I).
`The action of guanidine hydro-
`chloride on pseudoglobulin
`is specific
`in that
`the molecule splits as
`it unfolds.3
`There does not appear
`to exist any dimensional
`re-
`lation between
`the denatured whole molecules and the denatured
`the splitting of native protein
`halves such as has been observed with
`molecules (10). Simultaneous splitting and unfolding has also
`been observed in the denaturation of myogen by urea (11).
`The observed relation between the concentration of denaturing
`
`a See
`
`(2)
`
`foot-note
`
`8.
`
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`274
`
`Denaturation
`
`of Proteins.
`
`II
`
`denatured after
`irreversibly
`agent and the fraction of total protein
`relation
`found
`dialysis
`is in qualitative
`accord with the analogous
`for serum albumin,
`and may be interpreted
`on the basis of the
`statistical
`considerations
`discussed previously
`(2). Quantita-
`tively, however,
`these two sets of data differ from one another
`in
`that
`the limiting
`value of the fraction
`irreversibly
`denatured
`is
`64 per cent for pseudoglobulin when denatured by urea and 84 per
`cent when denatured by guanidine
`hydrochloride,
`as compared
`with 15 per cent for serum albumin when denatured by either
`agent.
`This
`indicates
`fundamental
`differences
`in
`the
`intrinsic
`structure of these two proteins.
`The difference in maximum
`yield
`of irreversibly
`denatured pseudoglobulin
`produced by urea and
`guanidine hydrochloride
`is probably due to their different modes
`of action.
`from 8 M urea solutions yields ma-
`denaturation
`“Reversible”
`terial of molecular
`size and shape similar
`to
`that of the native
`protein.
`Electrophoretic
`measurements
`on pseudoglobulin
`GI,
`denatured by 5 M urea, showed it
`to move with a
`“reversibly”
`single boundary on both
`the acid and alkaline
`side of the
`iso-
`electric point
`(12). The electrophoresis
`curves of the native and
`“reversibly”
`denatured material were practically
`indistinguishable
`from one another, except for differences
`in mobility.
`demands a
`The question of the true reversibility
`of denaturation
`critical examination
`of the data at hand.
`It was shown that
`the
`protein
`remaining
`in solution after
`isoelectric precipitation
`of the
`irreversibly
`denatured
`fraction was polydisperse.
`However,
`the
`spread
`in diffusion
`constants was relatively
`narrow
`(3.8
`to 4.6
`X lo-‘),
`suggesting
`that polydispersity was not due to incomplete
`separation of “reversed”
`denatured
`and denatured protein, but
`rather
`to a gradation
`in molecular
`size or shape of the “reversed”
`denatured
`protein
`itself.
`This assumption
`finds
`support
`in
`Poison’s diffusion measurements on whole serum globulin
`(13) ob-
`tained by precipitation
`with half saturated
`ammonium
`sulfate,
`where the spread in calculated diffusion constants
`is comparable
`to that observed here for the unfractionated
`“reversed” denatured
`pseudoglobulin.
`Further support comes from
`the observed solu-
`bility
`properties. When
`“reversible”
`denaturation
`was carried
`out with pseudoglobulin
`GII,
`precipitation
`of the
`“reversibly”
`denatured protein was found
`to occur over the region of 1.1 and
`sulfate, whereas the salting-out
`1.6 M ammonium
`region of the
`
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`
`Neurath, Cooper, and Erickson
`
`275
`
`Pseudo-
`to between 1.36 and 1.6 M.
`native material was confined
`started
`to ‘9eversible”
`clenaturation,
`globulin GI, when subjected
`to precipitate
`at an ammonium
`sulfate concentration
`of 0.8 M, as
`for the native protein, and continued up to
`compared with 1.1 M
`1.36 M. One may conclude, that “reversible” denaturation
`did not
`result
`in the restoration of a distinct molecular configuration,
`but
`rather
`in the
`formation
`of molecular
`entities of related
`intrinsic
`structures.
`This conclusion
`is also in accord with
`the
`ideas ex-
`pressed in a previous paper (12) concerning
`the continuous grada-
`tion
`in physical and chemical properties of the native globulins.
`A fraction approximating
`in properties
`the native material
`can be
`isolated
`from
`this mixture by subjecting
`it to fractional precipita-
`tion with salt under the same experimental
`conditions as have been
`used for the purification
`of the native protein.
`Further compara-
`tive studies of these fractions and of their
`immunological
`properties
`are under way.
`denatured protein
`irreversibly
`properties of the
`The solubility
`fractions of normal
`are similar
`to those observed for the euglobulin
`horse serum as obtained by the method of isoelectric precipitation
`(14). Like
`these euglobulin
`components,
`the relative
`viscosity
`of this material
`is much higher
`than
`that of the native pseudo-
`globulin
`(15). Attempts
`to
`identify
`the protein
`in
`terms of
`molecular weight or shape were impeded by
`its anomalous be-
`havior
`in respect to diffusion.
`
`to the
`to the Rockefeller Foundation,
`The authors are indebted
`Lederle Laboratories,
`Inc., and to the Duke University Research
`Council
`for support of this work.
`
`SUMMARY
`by urea follows
`The clenaturation of horse serum pseudoglobulin
`a similar pattern
`to that observed
`for serum albumin.
`The ap-
`parent molecular
`asymmetry,
`determined
`by viscosity memure-
`ments,
`increases with
`increasing concentrations
`of urea, whereas
`the diffusion constants decrease in proportion.
`The molecular
`weight remains unchanged during denaturation.
`The denaturing
`effects produced by guanicline hydrochloride
`ap-
`2 M guanicline hydrochloride
`depend on the concentration.
`pears to cause an aggregation of the protein molecules,
`the mean
`molecular weight being about
`twice that of the native protein.
`In
`
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`276
`
`Denaturation
`
`of Proteins.
`
`II
`
`into halves as they unfold,
`the protein molecules split
`3 M solution
`whereas a further
`increase
`in the guanidine hydrochloride
`concen-
`to 5.6 M, produces no additional
`changes
`in molecular
`size
`tration,
`or shape except
`that
`the solutions become monodisperse.
`Removal of the denaturing
`agent by dialysis causes a separation
`into
`two
`fractions which have been identified with
`“reversibly”
`and irreversibly
`denatured protein.
`The quantitative
`distribution
`between
`these two
`fractions
`is a function of the concentration
`of the
`denaturing
`agent.
`In equimolar
`concentrations,
`guanidine
`hy-
`drochloride
`leaves a larger
`fraction
`irreversibly
`denatured
`than
`does urea.
`pre-
`protein, purified by fractional
`denatured
`The “reversibly”
`cipitation with ammonium
`sulfate,
`resembles, but is not identical
`with,
`the native protein
`in respect
`to molecular
`size, shape, and
`electrophoretic
`properties.
`to solu-
`in respect
`resembles
`The irreversibly
`denatured protein
`as isolated by iso-
`bility and viscosity
`the euglobulin components
`The solutions ex-
`electric precipitation
`from normal horse serum.
`hibit a tendency
`for gel formation which
`is more pronounced
`on
`the acid side of the isoelectric point
`than on the alkaline side.
`
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`Page 12
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`
`THE DENATURATION OF PROTEINS
`AND ITS APPARENT REVERSAL: II.
`HORSE SERUM PSEUDOGLOBULIN
`Hans Neurath, Gerald R. Cooper and John O.
`Erickson
`1942, 142:265-276.
`
`J. Biol. Chem.  
`
`Access the most updated version of this article at
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