Viscosity of Aqueous Bovine Serum Albumin

By: Charles Erickson

Physical Chemistry Laboratory CHEM 4643 Section 2
October 5, 2006
Lab Partner: Eric P

Intrinsic viscosities were calculated for native and denatured aqueous bovine serum
albumin at 25C. A stock solution of 0.0300 g/ml BSA was prepared and diluted into
15/25ml, 10/25ml, and 7/25ml samples using 0.010M KCl solvent. A portion of the stock
solution was denatured and diluted into 30/50ml, 20/30ml, and 15/30ml samples using
0.010M KCl adjusted to pH 3. Viscosities of the eight solutions and KCl solvent were
found using a Ostwald type viscometer which required calibration. Calibration was
carried out with the use of water. The individual viscosities and their concentrations were
used to graphically determine the intrinsic viscosity. The viscometer constant was
determined to be 1.562*10^-4 3.134*10^-7 cm2/sec2 and the viscosity of the KCl
solution was 8.698*10^-4 8.995*10^-7 kg/sec*m. The native stock and denatured
stock intrinsic viscosities were 2.720.55 kg/m*sec and 8.301.30 kg/m*sec respectively.
Introduction

The purpose of this experiment was to measure the intrinsic viscosities of native and
denatured bovine serum albumin (BSA). The viscosity of a fluid measures its resistance
to flow under an applied shear stress [4]. Generally in fluid flow there is a velocity
difference across the thickness of the fluid that ranges from zero at the point of contact
with a wall or tube to a maximum at the surface of the fluid [3]. It is this velocity
difference across the width of the fluid that induceses shear stresses that resist the motion
of the fluid and give it its characteristic thickness. Fluids with a higher viscosity are
difficult to pour whereas fluids with a low viscosity are easily pourable.

Serum albumin is the most abundant protein in blood and is used to maintain its pH. Two
forms exist; native, the more compact form and denatured, the elongated form. To
measure the viscosity of both the native and denatured forms of BSA we used an Ostwald
viscometer. The viscometer was operated isothermally in a water bath at 25C. Using
the efflux time, or the time that a controlled volume of fluid passes between the upper and
lower marks on the viscometer and the Poiseuille Equation 1, we were able to calculate
the viscosity of our solutions.

/ = Bt (1)

Where is the viscosity, is the fluid density, B is the viscometer constant, and t is the
efflux time. Rearranging equation (1) and utilizing tabulated values for the viscosity and
density of water at 25C the viscometer constant can be calculated.

B = (/)/ t (2)

A potassium chloride solution was used as the solvent for this lab and its viscosity was
calculated using equation 3.

o = Bt (3)

Specific viscosity is the fractional increase in viscosity due to a solute, in the case of this
lab BSA.

sp = (- o) / o (4)

Taking the limit as c goes to zero of the ratio of the specific viscosity and the
concentration is defined as the intrinsic viscosity. Intrinsic viscosity is a measure of a
solutes contribution to the solutions viscosity.

= lim (sp/c) as c 0 (5)

For the purposes of this lab the intrinsic viscosity is estimated graphically by plotting the
specific viscosity divided by the concentration of the respective solution versus the same
concentration. The best fit line to this plot is solved for the intercept or the intrinsic
viscosity. Intrinsic viscosity is sensitive to the shape of the molecules in solution and can
provide rough estimates for various physical attributes such as the effective spherical
radius.

= 10NaRe3/(3M) (6)

Where Na is Avogadros number, Re is the effective spherical radius, and M is molecular

weight.

Alistofreagents,supplies,andadditionaldetailsareavailableinthelabhandout[1].

Experimental Methods

Severalreagentswereusedduringthissetofexperiments.Thefirstofwhichwas500ml
of0.010MKCl.Thissolutionwasusedasthesolventforallofthedilutionsinthelab
andwasalteredtodenaturetheBSAafterallofthenativeBSAsolutionswereprepared.
Thesecondwas100mlof0.0300gm/mlnativeBSAstocksolution.BSAwasinsolid
formandrequireddissolvingin80mloftheKClsolvent.Thestocksolutionwasstirred
untilnovisiblesolidBSAwaspresentandwasthenfilteredtoensurenosolidBSA
remained.Usingthenativestocksolutionthreedilutionswereprepared;15/25ml,
10/25ml,and7/25ml.A25mlsampleofthenativestockwasalsosetasideforviscosity
measurements.Next,40mloftheremainingnativeBSAstockwasdenaturedusing60ml
ofKClsolventwhichhadbecorrectedtopH3,anddropwiseadditionof1MHCl.
Oncethedenaturedstocksolutionwasprepared,threedilutionswereprepared;30/50ml,
20/30ml,and15/30ml.Theremainingvolumeofthedenaturedstocksolutionwasalso
retainedforviscositymeasurements.

Afterthesolutionsandtheirdilutionswerecreatedtheirviscositiesweremeasured.The
solutionsviscositiesweremeasuredbypipetting10mlofeachsolutionintothe
viscometerandmeasuringtheeffluxtimes.Theviscometerconstant,KClsolution,stock
BSAsolutions,andBSAdilutionsweredeterminedinthelistedorder.Allmeasurements
wereconductedisothermallyat25Cintheprovidedwaterbath.Asoutlinedabove,
literaturevaluesfortheviscosityanddensityofwaterarereadilyavailableandallowed
ustocalculatetheviscometerconstant.Severalrepetitionsofeachstepwerecompleted
toreduceourerror.Specialcarewastakentocleantheviscometerbetween
measurements.Afivestepwashingmethodthatinvolvedwater,DIwater,soap,and
acetonewaspreformedeachtimeanewsolutionwasintroducedintotheviscometer.

Adetaileddescriptionofthelabprocedures,equipment,andmeasurementdetailscanbe
foundinthelabhandout[1].
Results
Sample calculations for the various steps in this lab are hand written and attached at the
end.

Tabulated concentrations and efflux times for the native BSA and its dilutions are
presented in Table 1.

Table 1: Concentrations and Efflux Times for Native BSA Solutions

Native BSA solutions
c (g/mL) t1 (s) t2 (s) t3 (s)
Stock 0.02996 61 62.1 63
15/25 0.017976 59 59.1 59.2
10/25 0.011984 58.1 58 57.9
7/25 0.008389 57.2 57.3 57.1

Tabulated concentrations and efflux times for the denatured BSA and its dilutions are
presented in Table 2.

Table 2: Concentrations and Efflux Times for Denatured BSA Solutions

Denatured BSA solutions
c (g/mL) t1 (s) t2 (s) t3 (s)
Stock 0.011984 65.4 65.5 65.4
30/50 0.00719 60.9 60.8 60.6
20/50 0.004794 58.8 58.7 58.5
15/50 0.003595 57.9 58 57.7

The viscometer constant for each trial, the average viscometer constant, its standard
deviation, and efflux times are presented in Table 3. The constant was calculated using
Equation 2

Table 3: Viscometer Constant and Efflux Times

Viscometer Calibration
B (cm^2/sec^2) w (gm/cm*sec) w (gm/cm^3) t (sec)
1.564E-04 8.949E-03 0.9971 57.4
1.564E-04 8.949E-03 0.9971 57.4
1.558E-04 8.949E-03 0.9971 57.6
1.562E-04 Average
3.134E-07 Standard Deviation
The KCl solvent viscosity as determined using Equation 3, efflux times, and the accepted
density of solution are presented below in Table 4.

Table 4: KCl Solvent Viscosity and Efflux Times

KCl Solvent Viscosity
t
o (kg/m*sec) o (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) (sec)
8.709E-04 8.709E-03 1.562E-04 0.99751 55.9
8.693E-04 8.693E-03 1.562E-04 0.99751 55.8
8.693E-04 8.693E-03 1.562E-04 0.99751 55.8
8.698E-04 8.698E-03 Average
8.995E-07 8.995E-06 Standard Deviation

The native BSA solution viscosities as determined using Equation 3, efflux times, and the
accepted density of solution are presented in Table 5.

Table 5: Native BSA Solution Viscosities and Efflux Times

Native BSA solutions: Stock
(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)
9.503E-04 9.503E-03 1.562E-04 0.99751 61
9.675E-04 9.675E-03 1.562E-04 0.99751 62.1
9.815E-04 9.815E-03 1.562E-04 0.99751 63
9.664E-04 9.664E-03 Average
1.560E-05 1.560E-04 Standard Deviation

Native BSA solutions: 15/25

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)
9.192E-04 9.192E-03 1.562E-04 0.99751 59
9.207E-04 9.207E-03 1.562E-04 0.99751 59.1
9.223E-04 9.223E-03 1.562E-04 0.99751 59.2
9.207E-04 9.207E-03 Average
1.558E-06 1.558E-05 Standard Deviation

Native BSA solutions: 10/25

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)
9.051E-04 9.051E-03 1.562E-04 0.99751 58.1
9.036E-04 9.036E-03 1.562E-04 0.99751 58
9.020E-04 9.020E-03 1.562E-04 0.99751 57.9
9.036E-04 9.036E-03 Average
1.558E-06 1.558E-05 Standard Deviation

Native BSA solutions: 7/25

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)
8.911E-04 8.911E-03 1.562E-04 0.99751 57.2
8.927E-04 8.927E-03 1.562E-04 0.99751 57.3
8.896E-04 8.896E-03 1.562E-04 0.99751 57.1
8.911E-04 8.911E-03 Average
1.558E-06 1.558E-05 Standard Deviation
The denatured BSA solution viscosities as determined using Equation 3, efflux times, and
the accepted density of solution are presented in Table 6.

Table 6: Denatured BSA Solutions Viscosities and Efflux Times

Denatured BSA solutions: Stock
(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)
1.019E-03 1.019E-02 1.562E-04 0.99751 65.4
1.020E-03 1.020E-02 1.562E-04 0.99751 65.5
1.019E-03 1.019E-02 1.562E-04 0.99751 65.4
1.019E-03 1.019E-02 Average
8.995E-07 8.995E-06 Standard Deviation

Denatured BSA solutions: 30/50

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)
9.488E-04 9.488E-03 1.562E-04 0.99751 60.9
9.472E-04 9.472E-03 1.562E-04 0.99751 60.8
9.441E-04 9.441E-03 1.562E-04 0.99751 60.6
9.467E-04 9.467E-03 Average
2.380E-06 2.380E-05 Standard Deviation

Denatured BSA solutions: 20/50

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)
9.160E-04 9.160E-03 1.562E-04 0.99751 58.8
9.145E-04 9.145E-03 1.562E-04 0.99751 58.7
9.114E-04 9.114E-03 1.562E-04 0.99751 58.5
9.140E-04 9.140E-03 Average
2.380E-06 2.380E-05 Standard Deviation

Denatured BSA solutions: 15/50

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)
9.020E-04 9.020E-03 1.562E-04 0.99751 57.9
9.036E-04 9.036E-03 1.562E-04 0.99751 58
8.989E-04 8.989E-03 1.562E-04 0.99751 57.7
9.015E-04 9.015E-03 Average
2.380E-06 2.380E-05 Standard Deviation
The specific viscosities and data used for the graphical determination of the intrinsic
viscosities for all BSA solutions are presented in Table 7.

Table 7: Specific Viscosities and Data used for Graphical Determination of Intrinsic Viscosity
Intrinsic Viscosity Plotting Data and Specific Viscosities of BSA Solutions
C sp/C
(gm/ml) (ml/gm) sp
Native BSA solutions: Stock 0.02996 3.706 1.110E-01
Native BSA solutions: 15/25 0.017976 3.255 5.851E-02
Native BSA solutions: 10/25 0.011984 3.238 3.881E-02
Native BSA solutions: 7/25 0.008389 2.918 2.448E-02

Denatured BSA solutions: Stock 0.011984 14.347 1.719E-01

Denatured BSA solutions: 30/50 0.00719 12.288 8.836E-02
Denatured BSA solutions: 20/50 0.004794 10.586 5.075E-02
Denatured BSA solutions: 15/50 0.003595 10.130 3.642E-02

The graphical determination of intrinsic viscosity for native BSA is displayed in Figure 1.
A linear regression was also performed and is presented as Table 8.

Figure 1: Graphical Determination of Intrinsic Viscosity for Native BSA

Table 8: Linear Regression for Native BSA

Native BSA Linear Regression
Coefficients Standard Error Lower 95% Upper 95%
Intercept 2.7171 0.1283 2.1648 3.2692
X Variable 1 32.924 6.7760 3.7687 62.078
The graphical determination of intrinsic viscosity for denatured BSA is displayed in
Figure 2. A linear regression was also performed and is presented as Table 9.

Figure 2: Graphical Determination of Intrinsic Viscosity for Denatured BSA

Table 9: Linear Regression for Denatured BSA

Denatured BSA Linear Regression
Coefficients Standard Error Lower 95% Upper 95%
Intercept 8.3046 0.3015 7.0069 9.6022
X Variable 1 512.76 39.666 342.08 683.42

The intrinsic viscosities for the native and denatured BSA and the effective spherical
radius are presented in Table 10.

Table 10: Intrinsic Viscosities and Effective Spherical Radius

(ml/gm) Re (nm)
Native 2.7171 3.06
Denatured 8.3046 4.44
Discussion

The purpose of this lab was to determine the intrinsic viscosities of native and denature
bovine serum albumin. Values obtained in lab, error estimates and literature values [2]
can be found in Table 11.

Table 11: Comparison of Lab Values and Literature Values

Lab Results Literature2
Students Accepted
(ml/gm) 95% CI (ml/gm) 95% CI (ml/gm)
Native 2.72 0.55 3.4 0.6 3.7
Denatured 8.30 1.30 11 4 11

Our results are similar to those of the students found in [2], but differ slightly from the
accepted values. This can be demonstrated because the error associated with each value
causes their ranges to overlap indicating no difference between means. This is best
demonstrated in graphical form. Figure 3 displays our values, the students values, and
the accepted values. Overall we showed that the native BSA has a lower viscosity than
the denatured BSA.

Figure 3: Comparison of Means for Native and Denatured BSA

In addition to the intrinsic viscosities the effective spherical radius was also calculated.
We showed that the effective spherical radius for the denatured BSA (4.44nm) was larger
than the native BSA (3.06nm). This agrees well with the results of the viscosity tests
because the denatured solution had a higher viscosity due to its larger molecule size.
The calibration of the viscometer with water resulted in a constant of 1.562*10^-4
3.134*10^-7 cm2/sec2.Thisvalueiscrucialtotherestofthelabandthusseveral
repetitionsoftheeffluxtimeswereconducted.Theerrorassociatedwiththeconstantis
threeordersofmagnitudesmallerwhichisgood.

TheviscosityoftheKCLwasalsocrucialtothesuccessofthelabresults.Theaverage
valuewas8.698*10^-4 8.995*10^-7 kg/sec*m. Thisvalueiscrucialtotherestofthe
labandthusseveralrepetitionsoftheeffluxtimeswereconducted.Theerrorassociated
withtheconstantisthreeordersofmagnitudesmallerwhichisgood.

Some of the differences between our data and the accepted values and those of other
students could stem from a number of sources. This lab is very sensitive to temperature,
timing, and concentration. The use of the water bath allowed us to control the
environment in which we ran the viscosity tests. However, the water temperature did
change due to convective losses to the room and conductive losses to the beakers and
viscometer we place in the bath. If the bath were insulated there may be better
temperature control, however this would make it difficult to observe the viscometer.
Secondly, the measurement of the efflux times is done by hand. This introduces a lot of
human error. Finally, the change in concentration of the stock solution due to filtering
may also affect the end results. When filtering the BSA solution it was observed that it
foams easily and that not all of the BSA dissolves into solution. There are no doubt
losses in this step of the procedure.

Finally it should be noted that during the course of the two week lab we encountered
troubles. We had several human errors that ultimately left us with unusable data. As a
result, I spoke with Dr. Siders and was supplied with data to conduct the analysis. The
report and data analysis were conducted as if the data was my own to avoid confusing
wording and nomenclature. In the future we need to pay more attention to unit
conversions and what glassware is supplied with the lab.
References

[1] University of Minnesota Duluth Chemistry Department Lab Manual,

Viscosity of Aqueous Bovine Serum Albumin, 2006

[2] Richards, J. L. Viscosity and the Shapes of Macromolecules: A Physical

Chemistry Experiment Using Molecular-Level Models in the
Interpretation of Macroscopic Data Obtained from Simple Measurements.
Journal of Chemical Education 1993, 70(8), 685-689.

[3] Engel, Thomas and Philip Reid, Thermodynamics, Statistical

Thermodynamics, & Kinetics, Benjamin Cummings, 2005.

[4] James O. Wilkes, Fluid Mechanics for Chemical Engineers, Stacy G.

Bike, 1999