Handbook of Size Exclusion Chromatography and Related ...

water-soluble and detergent-soluble membrane proteins. All the points for the
water-soluble proteins lie on a sigmoid curve (except fibrinogen, which has
different behavior as aresult of its asymmetrical shape). The membrane proteins
clearly fall outside the calibration curve for water-soluble proteins, so that the
Stokes radii estimated from this curve are high by 10–30%.
HimmelandSquire(84)foundsignificantimprovementinthedetermination
of protein molecular weight using denaturing conditions. Their study reconciles
the size parameters of proteins and random coils by determining F(v) in Eq. (17):
F(v) ¼ V1=3 E
V 1=3
T
V 1=3
i
V 1=3
i
MuchlesserrorforthemolecularweightdeterminationisfoundwhenplottingF(v)
!
(17)
vs. MW 1/3 than KD vs. logMW, RS vs. MW 1/3 ,or K 1=3
D vs. MW1/3 .Tarvers and
Church (91), working with TSKgel G3000SW columns, utilized both native and
denaturedproteinstocompareplotsofF(v)vs.MW 1/3 ,RSvs.erf (1 F(v)),andRS
vs.erf (1 KD)andconfirmedplotsofF(v) vs.MW 1/3 providedabetterestimate
of protein molecular weight. The method of Himmel and Squire (for example,
F(v) vs. MW 1/3 )has been used to produce linear curves with native proteins
(92–94), denatured proteins (95), and,independently,globular proteins (96).
Denaturing gel filtration with 0.1% sodium dodecyl sulfate (SDS) or 6M
guanidine hydrochloride results in better resolution, increased accuracy,and an
extended linear range. This provides asimple, rapid, and sensitive means of
separating protein mixtures and determining protein molecular weights that
deviateonly5–7%fromreportedvaluesmeasuredbygelfiltration,sedimentation
equilibrium, or SDS–polyacrylamide gel electrophoresis (97). On TSKgel
G3000SW(Fig.13),thelinear partofthecalibration curvefor proteins denatured
inguanidinehydrochlorideextendsfrommolecular weight9000to43,000.Using
the same column, the calibration curve for SDS-denatured proteins is linear from
9000 to93,000, andnondenaturing conditionsprovide alinear curvefrom 30,000
to 93,000 with no resolution below 30,000. Similar work by Kato (58) provided
the optimum separation ranges presented in Table 7. Good agreement on protein
behavior was seen between the various studies for G3000SW columns.
4 SECONDARY RETENTION
Schmidt et al. (98) showed how retention volumes of globular proteins varied on
silica-based diol bonded phase columns depending on the pH and ionic strength of
the mobile phase and their effective charge. Because most proteins elute within the
interstitial pore volume, size exclusion is the dominant effect; other possible
mechanisms are secondary order effects (99). Pfankoch et al. (33) investigated
© 2004 by Marcel Dekker, Inc.