`
`CSL EXHIBIT 1049
`
`Page 1 of 7
`
`CSL V. Shire
`
`Page 1 of 7
`
`CSL EXHIBIT 1049
`CSL v. Shire
`
`

`

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`Volume 16 Number 1
`February 1994
`IJBMDR 16(1)
`1—56 (1994)
`ISSN 0141 —8130
`
`Biological
`Macromolecules
`
`STRUCTURE, FUNCTION AND INTERACTIONS
`
`Close mutual contacts of the amino groups in DNA
`J. Sponer and J. KypI
`Onset of the fully extended conformation in (oMe)Leu derivatives and
`short peptides
`C. Tania/o, M. Pantano, F. Formaggio, M. Crisma, G. M. Banana, A. Aubry,
`D. Bayeul, A. Dautant, W. H. J. Boesten, H. E. SchaemakerandJ. Kamphuis
`
`A study of enzymic degradation of a macromolecular substrate. poly[N°-(2-
`hydroxyethyl)-L-glutamine]. by gel permeation chromatography and kinetic
`modelling
`J. Pyte/a, J. Jakes and F. Rypaéek
`
`Heparin binding to monodisperse plasma fibronectin induces aggregation
`without large-scale changes in conformation in solution
`L. Vui/lard, D. J. S. Hu/mes, I. F. Purdom and A. Miller
`
`Collagen organization in an oriented fibrous capsule
`B. Brodsky and J. A. M. Ramshaw
`
`Viscometric study of human, bovine, equine and ovine haemoglobin in
`aqueous solution
`K. Monkos
`
`Limited proteolysis of fl-Iactoglobulin using thermolysin. Effects of calcium
`on the outcome of proteolysis
`E. Dufour. M. Dalga/arrondo and T. Heart/e
`
`Enzymatic degradation of chitins and partially deacetylated chitins
`Y. Shigemasa. K. Saito, H. Sashiwa and H. Saimoto
`Book review
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`IBC Notes for authors
`
`Int. J. Biol Macromol. 1994 Volume 16 Number 1
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`Page 2 of 7
`
`

`

`Viscometric study of human, bovine,
`equine and ovine haemoglobin in
`aqueous solution
`
`K. Monkos
`Department of Biophysics, Silesian Medical Academy, H. Jordana 19, 41-808 Zabrze 8,
`Po/and
`
`Received 28 June 1993; revised 28 September 1993
`
`This paper presents the results of viscosity determinations on aqueous solutions of several
`mammalian haemoglobins at an extremely wide range of concentrations. Rheological
`quantities such as the intrinsic viscosity and Huggins coefficient were calculated on the
`basis of the modified Mooney's formula. Using the dimensionless parameter c[17] , the
`existence of three characteristic ranges of concentrations was shown . By applying Lefebvre's
`formula for the relative viscosity in the semi-dilute regime, the Mark-Houvink exponent was
`evaluated.
`
`Keywords: viscosity; Huggins coefficient; Mark- Houvink exponent
`
`Viscometric measurements, as a convenient experimental
`tool, are still extensively used in many investigations of
`both synthetic polymers and biological macromolecules
`in solution 1 - 7 . In biological systems, proteins (when
`present as solutions) are found in most cases al rather
`high concentrations. This is especially the case for
`baemoglobin (Hb), which is present in erythrocytes at
`the extremely high concentration of 5.4 mmol 1- 1
`(Ref. 8). Our knowledge of the properties of proteins in
`concentrated solutions is still limited, and one of the
`reasons for this is that the choice of methods available
`ismuch more limited than in the case of dilute solutions.
`Only viscometry, which is a simple and convenient
`method, allows the study of molecular sizes and shapes
`as well as interactions of proteins in concentrated media.
`Viscometric measurements alone, or in conjunction with
`other methods such as dielectric or electron paramagnetic
`resonance spectroscopy, have been used in investigations
`14
`ofhuman haemoglobin in solutions 9
`. However, as far
`-
`as we know, very little attention has been paid to the
`viscometric study of other mammalian haemoglobins.
`ln the present work, we report results on the viscosity
`of solutions of human, bovine, equine and ovine
`baemoglobin over a
`large range of concentrations
`extending from the dilute regime to concentrations higher
`than in physiological conditions. The dependence of
`viscosity on concentration in terms of the modified
`Mooney's equation is discussed. The Huggins coefficient
`and the exponent of the Mark- Houvink equation were
`determined for ail investigated haemoglobins.
`
`Experimental
`Materials
`Human blood was obtained from healthy, haemato-
`
`logically normal adult volunteers via venepuncture into
`heparin. Bovine, equine and ovine blood samples
`were taken in the same way. The fresh erythrocytes
`were washed several times with 0.9% NaCI solution.
`Membrane-free Hb solutions were prepared by haemolysis
`with water, followed by high-speed centrifugation. The
`pH values of such prepared samples were as follows:
`human Hb, pH 7; bovine Hb, pH 7.3; ovine Hb, pH 7.4;
`equine Hb, pH 7.7. These values changed insignificantly
`during the dilution of the solutions. The samples were
`stored at 4°C untiljust prior to viscometry measurements,
`when they were warmed to 25°C.
`
`Viscometry
`Capillary viscosity measurements were conducted
`using an Ubbelohde microviscometer placed
`in a
`waterbath controlled thermostatically at 25 ± 0.1 °C. The
`same viscometer was used for ail measurements and was
`mounted so that it always occupied precisely the same
`position in the bath. Flow times were recorded to within
`0.1 s. Solutions were temperature-equilibrated and passed
`through
`the capillary viscometer before any
`once
`measurements were made. Five
`to
`ten flow-time
`measurements were made on each concentration. The
`relative viscosity 17r was measured, where 17r = 17/17 0 and
`17 and 17 0 are the viscosities of the solution and the sol vent,
`respectively.
`Solution densities and haemoglobin concentrations
`were measured by weighing, as described previously 7.
`The relative viscosities of the haemoglobin solutions were
`measured for concentrations from several g 1- 1 up to
`~ 525 g 1- 1 for human Hb, ~ 500 g 1- 1 for equine Hb
`and ovine Hb, and ~490 g 1- 1 for bovine Hb. The results
`are shown in Figure 1.
`
`0141-8130/94/01003 l - 05
`1994 Butterworth-Heinemann Limited
`
`Int. J. Biol. Macromol., 1994 Volume 16 Number 1 31
`
`Page 3 of 7
`
`

`

`Viscometric study of mammalian haemoglobins: K. Monkos
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`100
`
`200
`
`300
`dg/ 1]
`Figure 1 Plot of the relative viscosity ~' versus concentration c for
`human (_..), ovine ( x), equine (• ) and bovine haemoglobin (6); the
`curves show the fit obtained by using equation (3) with parameters A
`and B from Table 1
`
`400
`
`500
`
`Results and discussion
`Mooney's approximation
`Despite substantial efforts 15, a useful theory for the
`viscosity of moderately concentrated and concentrated
`solutions does not yet exist. Much effort bas therefore
`been devoted
`to a search for empirical functional
`representations
`incorporating a wide concentration
`16
`range 12
`. However, as bas been shown in our earlier
`•
`work 1 7
`, in the case of aqueous solutions of globular
`proteins, the most useful functional form describing the
`dependence of relative viscosity on concentration is that
`of Mooney 18
`:
`
`(1)
`
`11, = exp[ l ~~9' J
`where q, is the volume fraction of the dissolved particles,
`S denotes the shape parameter and K is a self-crowding
`factor. The volume fraction q, = NA Ve/ M where NA, V
`and M are Avogadro's number, the volume of one
`dissolved particle and the molecular weight, respectively.
`The solute concentration c is in g 1- 1
`. In bis original
`work 18
`, Mooney obtained equation (1) for spherical
`particles for which S = 2.5, so that, in the limit q, ....... 0,
`the equation yields the expression developed by Einstein:
`IJ, = 1 + 2.5q,. In the case of particles of arbitrary shape,
`S should exceed 2.5.
`It is known that the volume ofhydrodynamic particles
`may include a shell of water of hydration. Because the
`shell may change with concentration, it is difficult to
`evaluate accurately the value of q, as a fonction of protein
`concentration. This problem was circumvented by Ross
`and Minton 9
`. They generalized Mooney's equation to
`the form:
`
`'7r = exp
`[
`
`[17]c
`
`]
`
`1 -
`
`; [17]c
`
`(2)
`
`32
`
`Int. J. Bio!. Macromol., 1994 Volume 16 Number l
`
`where
`[ J 1.
`'7sp
`17 = 1m-
`c-o C
`is the intrinsic viscosity and IJsp = 17, - 1 is the specific
`viscosity. There is only one adjustable parameter (K /S)
`in equation (2). For two sets of data for human
`haemoglobin, the best fit of the above formula to the
`experimental points was obtained for K /S = 0.4 and 0.42.
`However, as seen in Figure 1 in Ref. 9, the fit is not the
`best one, especially m the moderately concentrated
`region.
`The problem can be treated in another way. The
`volume fraction q, can be rewritten as q, = rxc, where
`rx =NA V/M, and then equation (1) takes the form:
`
`IJr = exp[~]
`1- Be
`
`(3)
`
`where A = rxS and B = rxK are two adjustable parameters
`and the ratio B/ A = K/S. Mooney's relation given in the
`above form bas two merits: (i) it is not necessary to know
`the intrinsic viscosity, and (ii) by fitting of the two
`parameters A and B, the equation gives good approximation
`to experimental values over the whole range of concentra(cid:173)
`tions and the ratio K/S can be obtained as well.
`Mooney's equation bas been fitted in this way to the
`experimental values for ail haemoglobin solutions
`investigated. As seen in Figure 1, a good fit over the
`whole range of concentrations was obtained. The
`adjustable parameters for ail samples are shown in Table 1.
`The value of K /S = 0.432 obtained for human haemo(cid:173)
`globin is in good agreement with that of Ross and
`Minton. However, the most interesting parameters are
`the absolute values of S and K in equation (1). Sorne
`indications about these parameters can be obtained in
`the following way. As is known from crystallographic
`studies, human haemoglobin is a spheroid with main axes
`64 x 5 5 x 50 Â 19 and molecular weigh t 68 000. Let us
`suppose that there is no hydration shell around the
`haemoglobin molecules. In this case, the volume fraction
`q, can be calculated for ail concentrations, and equation (1)
`can be fitted
`to
`the experimental points with two
`parameters S and K. Such a procedure gives S = 3.45
`and K = 1.491 for our experimental data. Tanford 20 bas
`calculated that the value of S for human haemoglobin
`should lie between 2.5 and 4.8. This allows indirect
`calculation of the self-crowding factor K, using the
`experimental values of K /S. The value of K/S = 0.4
`obtained by Ross and Minton 9 gives, in this case, a value
`of K in the range between 1 and 1.92. For our value of
`K /S = 0.432, K is in the range of 1.08 to 2.07. Our values
`
`Table l Parameters of the haemoglobin samples obtained from the
`fit of Mooney's relation to the experimental points (Figure 1) and froni
`equations (6) and (7)
`
`Haemoglobin
`
`Human
`
`Bovine
`
`Equine
`
`A (mlg - 1)
`B (mlg - 1
`)
`J!._K
`A -
`S
`k,
`k2
`k2/kî
`
`2.77
`1.2
`0.432
`0.932
`0.786
`0.904
`
`4.37
`1.03
`0.236
`0.736
`0.458
`0.846
`
`4.22
`0.96
`0.228
`0.728
`0.447
`0.843
`
`O vine
`
`3.4
`1.06
`0.312
`0.812
`0.576
`0.874
`
`Page 4 of 7
`
`

`

`of s and K lie nearly in the middle of these ranges and
`tbey probably are very close to true values for human
`baemoglobin. However, such evaluation is only possible
`in the case of molecules of known sizes. I t is worth noting
`tbat Tanford 20
`, using independent measures of the
`amount of water of hydration, has also estimated that
`the most probable value of Sin this case lies between 3.7
`and 3.9. Because the ratio of K /S should not depend on the
`amount of water of hydration, one can use it to calculate
`K. For K/S = 0.4, it gives K in the range of 1.48 to 1.56,
`and for K/S = 0.432, K is in the range of 1.598 to 1.68.
`As seen from Table 1, substantial differences exist
`in the values of K /S for the investigated samples. This
`indicates that different mammalian haemoglobins do not
`bave the same shape in solution and/ or that they interact
`with the solvent in a somewhat different manner.
`
`Jntrinsic viscosity and the Huggins coefficient
`At
`low concentrations, the relation between the
`solution viscosity and the concentration may be expressed
`by the polynominal 16:
`
`t/sp = [17] + k1[11] 2c + k2[17] 3
`
`C2 + ...
`
`(4)
`
`C
`where the dimensionless parameter k1 is the Huggins
`coefficient. The simplest procedure for treating viscosity
`data consists of plotting the l7sp/c against concentration,
`extrapolating it to the intercept (equal to [17]) and
`obtaining the coefficient k 1 from the corresponding slope.
`However, as was pointed out in Ref. 16, even if l7sp < 0.7,
`the concentration dependence of 11.p/c is curved, so that
`linear extrapolation gives a serious error in [17] and k 1 .
`The problem can be solved for solutions for which the
`conditions of Mooney's formula are fulfilled. Mooney's
`equations (1) or (3) can be expanded in the power series
`of concentration. Limiting to the second-order term, an
`identical expansion to that in equation (4) can be obtained
`from:
`['/] = a.S = A
`
`(5)
`
`and
`
`(6)
`
`(7)
`
`k, = t( 2: + 1)
`1( K2
`k2 = - 6- + 6 - + 1
`)
`K
`S2
`S
`6
`The
`the Huggins coefficient
`intrinsic viscosity and
`obtained on the basis of equations (5) and (6), for ail our
`haemoglobin samples, are shown in Table 1. The results
`of theoretical calculations for rigid, non-interpenetrating
`spheres have given a range of numerical values of k 1
`16 .
`It . seems
`that our results for equine, bovine and
`;~me haemoglobin are quite consistent with results of
`irn~mann 21 (k 1 = 0.76) and with the precise results
`obtamed for the Gaussian random coi! chain by Freed
`and ~dwards 22 (k 1 = 0.7574). Surprisingly enough, the
`Huggms coefficient value for human haemoglobin is close
`~ the value k 1 = 0.894 obtained by Peterson and
`1xman 23 in a mode! of penetrable spheres.
`There are no theoretical estimations of the second
`COe~cient k2 in equation (4). However, the theory 24
`~r~d1cts that, for rigid, non-interpenetrating spheres, the
`atJo k2/kî should equal 1. The values of k2 calculated
`on the basis of equation (7), as well as the ratios k2/kî,
`
`Viscometric study of mammalian haemog!obins: K. Monkos
`
`0.7
`
`0.6
`
`0.5
`
`0.6
`
`0.7
`
`0.5
`
`Figure 2 Correlation of coefficients k1 and k2. Experimental data:
`human (A), ovine ( x ), equine (• ) and bovine haemoglobin (.0.); the
`stra1ght lme 1s plotted according to equalion (8)
`
`are given in Table 1. For ail investigated haemoglobins,
`k 2/kf # 1. However, as is shown in Figure 2, the plot of
`k2 versus kf is linear and the following analytical relation
`is fulfilled (with correlation coefficient 0.999):
`
`k2 = kf - 0.0834
`
`(8)
`
`lt is worth noting that Maron and Reznik 25
`, on
`the basis of experiments with polystyrene and poly(cid:173)
`(methylmethacrylate), obtained a similar relation with a
`numerical value of 0.09. This suggests that equation (8)
`has quite a general character and is correct for different
`sorts of molecules.
`
`Three ranges of concentrations and determination of the
`M ark-H ouvink exponent
`The usual method of generalization of experimental
`results for different polymer systems consists of using
`reduced variables. In the case of the viscosity-concentration
`relation, this parameter is a dimensionless quantity
`[17]c26. The dependence of the specific viscosity on [17]c
`in a log- log plot exhibits classical behaviour for ail our
`samples, with transitions from dilute to semi-dilute
`solution at concentration c*, and from semi-dilute
`to concentrated solution at concentration c**. Such
`behaviour has been observed for cellulose derivatives 27
`,
`citrus pectins 28 and some globular proteins in random
`coi! conformation 29
`. In Figure 3, the master curve for
`bovine haemoglobin is shown. The master curves have
`the same form for the other haemoglobins. The parameters
`describing the curves are shown in Table 2. The boundary
`concentrations c* and c** are nearly superimposed,
`especially for equine, bovine and ovine haemoglobins.
`In the dilute region (c[17] < c*[17]), the molecular
`dimension is not perturbed by the other molecules and
`the average hydrodynamic volume of the molecule is the
`same as for infinite dilution. As is seen in Table 1, the
`slopes for ail investigated samples are nearly identical in
`this range. lt is worth noting that the slopes in the dilute
`domain are in the range of 1.1- 1.4 for quite different
`27
`28
`sorts of molecules 1
`•
`•
`.
`As was shown by Lefebvre 29
`, in the semi-dilute region,
`the following equation for the relative viscosity is fulfilled:
`C )1/2a
`ln 17, = 2a[17Jc* c*
`(
`
`- (2a -
`
`l)c*[17]
`
`(9)
`
`Int. J. Bio!. Macromol., 1994 Volume 16 Number 1 33
`
`Page 5 of 7
`
`

`

`Viscometric study of mammalian haemoglobins: K. Monkos
`
`1.5
`
`1.0
`a. "' ~
`B10.5
`
`0.0
`
`-0.5
`
`-1.0
`
`• • •
`• •
`•
`
`•
`Îc*
`
`-1
`
`- 0. 5
`
`0
`
`log ["l,k
`
`0. 5
`
`Figure 3 Specific viscosity as a functi on of c(~] in a log- log plot fo r
`bovine haemoglobin; straight lines show different slopes in dilute
`(c < c*) and concentrated (c > c**) regions
`
`where a is the Mark- Houvink exponent. Figure 4 shows
`the experimental points for bovine haemoglobin and the
`taking c* and a as
`curve resulting from
`the fit,
`adjustable parameters. The values of c* obtained by this
`procedure are in good agreement with
`the values
`determined from the master curve of Figure 3. The values
`of a (Table 2) are nearly the same, except for human
`haemoglobin. The Mark- Houvink exponent for flexible
`polymers is in the range of 0.5- 1 (Ref. 30). However,
`a= 0 in the case of bard spherical particles, and a= 1.7
`for hard long rods. The Mark- Houvink exponent values
`listed in Table 2 indicate that ail haemoglobins studied
`here behave as hard quasi-spherical particles, in agreement
`with the mode! proposed for human haemoglobin by Ross
`13
`31
`and Minton 9
`. It is important to add that the
`•
`•
`Lefebvre equation was originally applied to zero shear
`rate data. In our case, for concentrations close to c**,
`
`100
`
`200
`
`300 dg/ lJ
`
`Figure 4 Plot of the relative viscosity versus concentratio_n in a
`log- normal plot in a semi-dilute region. (• ) expenmental points _for
`bovine haemoglobin; the curves show the fit obtamed by usmg equallon
`(9)
`
`34
`
`Int. J. Bio!. Macromol., 1994 Volume 16 Number 1
`
`Table 2 Parameters of the haemoglobin samples obtained from the
`fit of the curves in Figures 3 a nd 4 and from equa tion (9)
`
`Haemoglobin
`
`Human
`
`Bovine
`
`Equine
`
`a
`c* (g 1- 1)
`c** (g l - 1)
`c* (11]
`c** (11]
`Slopes
`C < c*
`C > c**
`
`0.3
`76.3
`394
`0.21
`1.09
`
`1.1
`7.57
`
`0.338
`66.5
`375
`0.29
`1.64
`
`1.13
`7.02
`
`0.348
`64.9
`376
`0.27
`1.59
`
`1.1 2
`6.47
`
`Ovine
`
`0.333
`67.2
`375
`0.23
`1.28
`
`1.1
`6.05
`
`the shear rate was about 100 s - 1
`• However, as was quite
`recently shown by Müller et al. 14 for shear rates ranges
`from 1 to 200 s - 1
`, human haemoglobin solutions exhibit
`Newtonian behaviour up to a concentration of at least
`450 g 1- 1
`. This indicates that application of the Lefebvre
`equation in our case is justified and th at the Mark(cid:173)
`Houvink exponent has its usual meaning.
`In the concentrated region (c[1'/] > c**[17]), the elfects
`of entanglements become important. As was shown by
`Axel os et al. 2 8 for citrus pectins, which are relatively
`flexible polymers, the slope in this region is 3.4. A higher
`value (about 5) was obtained by Castelain et al. 2 7 fo r a
`non-rigid molecule of hydroxyethylcellulose. The values
`Jisted in Table 2 suggest that the highest value of slope
`in this region occurs for stilf molecules. This is in
`agreement with the results of Ref. 32, where the a uthors
`showed that the slope should be approximately 8 for stiff
`chained molecules. It is interesting to note that the
`second critical concentration c** for ail investigated
`haemoglobins is only slightly higher than the concentration
`of the haemoglobin in erythrocytes, which is about
`5.4 mmol J - 1 or 367 g 1- 1 . This may ex plain why the
`concentration of haemoglobin in erythrocytes is not
`higher, i.e. the haemoglobin concentration achieves the
`highest value at which entanglements are not yet present.
`In other words, haemoglobin molecules in erythrocytes
`achieve the highest concentration at which they can move
`relatively freely with minimal frictional interaction.
`
`Conclusions
`The viscosity of mammalian haemoglobin solutions over
`a wide range of concentrations at pH values near the
`isoelectric point may be quantitatively described by t~e
`modified Mooney's equation (equation (3)). On the bas1s
`of Mooney's asymptotic form for small concentrations,
`the intrinsic viscosity and the Huggins coefficient maY
`be calculated. The Huggins coefficient k 1 and the second
`coefficient of expansion k 2 are connected by equation (8),
`which seems to have quite a general form. The values for
`the Mark- Houvink exponent confirm that ail investigated
`haemoglobins behave as bard quasi-spherical particles.
`The values for the second critical concentration c**
`suggest that mammalian haemoglobin concentration in
`erythrocytes achieves the optimum value. Despite !he
`similarities, substantial dilferences exist between spec1es,
`especially for K /S and k1 values. This indicates that e~ch
`mammalian haemoglobin in solution should be stud1ed
`in detail separately.
`
`Page 6 of 7
`
`

`

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`lnt. J. Bio!. Macromol., 1994 Volume 16 Number l 35
`
`Page 7 of 7
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