Figure 8.3.5 Calibration of GF column for determination of protein size. A K 26/70 column packed with Sephadex G-200 SF is used. (A) Chromatogram obtained for six size standards: 1, catalase; 2, aldolase; 3, bovine serum albumin; 4, ovalbumin; 5, chymotrypsinogen A; 6, ribonuclease A. (B) Calibration curve obtained by plotting the logarithms of the molecular radii (log10R) of the standards (xaxis) versus the elution volumes for peaks 1, 2, 4, 5, and 6 from panel A (filled circles). The curve was used for evaluating the size of bovine serum albumin (filled square; elution volume corresponds to peak 3 in panel A), giving an apparent size of 34.8 A. The excellent agreement with the nominal molecular size value of 35.5 A may be incidental. Repeating the procedure for peak 4 in panel A showed that a deviation of ~3 A from the nominal value may be expected for proteins of globular shape. Panel A reproduced from Pharmacia Biotech (1991) with permission from the publisher.

calibrate the column is by using the hydrodynamic volume, Vh. This is calculated according to the equation Vh = (n)(Mr)/Nv, where n is the intrinsic viscosity, Mr is the relative molecular mass, N is Avogadro's number (6.02 x 1023 molecules per mole), and v is a shape factor, which for a spherical solute is 2.5.

It is wise to check the accuracy of the calibration by deleting one point at a time from the calibration data set and calculating the size or mass of the protein represented by that point from the calibration curve obtained with the remaining data. The average absolute difference from the nominal value indicates the accuracy of the calibration (see Fig. 8.3.5).

For calibration with denatured standards

1b. Denature calibration standards according to a standard denaturing protocol.

2b. Carry out steps 1a and 2a above, using a column equilibrated in 6 M guanidine hydrochloride and using 6 M guanidine hydrochloride as the GF buffer in the cross-referenced steps of Basic Protocol 1.

Where denatured standards are used, denaturing conditions must be maintained in chromatography of the sample (see Basic Protocol 3).

For calibration with nonprotein polymer standards

1c. Using an in-line refractive index detector, run system until a stable baseline is obtained. Chromatograph a number of dextran calibration standards (see Basic Protocol 1, steps 20 to 23) that will give calibration points surrounding the protein under study, to obtain their elution volumes or distribution coefficients.

The proper number and size of calibration standards to be run may be quickly found by running first the unknown sample and then the necessary calibration samples that will be eluted close to the unknown protein.

Apply each standard individually or as a mixture of two dextrans of high and low molecular mass.

Using a two-channel recorder set at two different sensitivities may prevent the peak from running off scale.

2c. Plot the elution volume (Vr) or distribution coefficient (KD) versus the logarithm of the viscosity radius of dextran (log10Rvis).

The viscosity radius of dextran, Rvis, is related to the molecular mass (M) according to the equation Rvis = 0.271 x M0-498. Because dextran is apolydispersepolymer, the value of M used here is the molecular mass of dextran that corresponds to the peak apex, sometimes denoted Mp.

When two different detectors are used for calibration and chromatography of the sample— e.g., a refractive index detector for dextran standards and a UV detector for the protein sample—the difference in volume between the two detectors must be taken into account in calculation of elution volume.

Another procedure where the entire separation range of interest was calibrated in one run, using an integral calibration method, has been described (Hagel and Andersson, 1991; Hagel, 1993). Unfortunately, this simple and powerful method cannot yet be performed on routine basis because it requires data for the molecular mass distribution of the sample, and this information is, at the moment, not readily available in most cases.




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