Molecules are separated by size. Small molecules enter pores of stationary phase (b), large molecules do not (a) Larger molecules elute first

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2 Molecules are separated by size Small molecules enter pores of stationary phase (b), large molecules do not (a) Larger molecules elute first

3 Types of gels for open column, preparative scale GPC (i) Bio-Gel P: Crosslinked polyacrylamide (BioRad)

4 Types of gels for open column, preparative scale GPC (ii) Structure of Sephadex, a crosslinked dextran sold by Pharmacia Fine Chemicals (Piscataway, NJ)

5 BioGel-P and Sephadex are also used for affinity chromatography: Gel Permeation Affinity Y = antibody, covalently attached to gel = antigen, protein of interest to isolate

6 D.C. Harris, Quantitative Analysis, 8th ed., WH Freeman & co., 2010

7 Molecular weight determination GPC mainly used to separate molecules with significant differences in MW Fore each stationary phase: log relation between MW and elution volume or K AV Unknowns compared to standards Warning: proteins of same MW may fold in different shapes (use high ionic strength to avoid this problem)

8 Molecular mass calibration for polystyrene on Beckman μspherogel molecular Exclusion column (0.77 x 30 cm). Pore sizes are indicated on each data set. D.C. Harris, Quantitative Analysis, 8th ed., WH Freeman & co., 2010

9 Book Title: Proteins Series: Methods in Molecular Biology Volume: 1 Pub. Date: Nov Page Range: 5-12 DOI: / :5

10 Developments in gel permeation supports for HPLC have made rapid separations possible. Gel permeation HPLC has become a widely used technique for MW measurements. It takes less time than SDS-PAGE, allows easier quantitation and recovery of separated proteins. The volume accessible to a protein in gel permeation supports depends on its size and shape. Sample proteins must have the same shape as proteins used for calibration. In the presence of denaturants, such as 6M guanidine hydrochloride, all reduced proteins adopt a linear random coil conformation, whose molecular radius is proportional to molecular weight.

11 A wide range of sensitivities can be covered, from femtomolar to nanomolar amounts. Because of the high absorbance of guanidine hydrochloride at < 220 nm, the eluate cannot be monitored at this wavelength, therefore proteins in the nanomolar range are detected by absorbance at 280 nm. Up to approximately 50 nmol can be separated on standard size columns (7.5 x 600 mm). Amounts greater than this reduce the resolution of the separation. Larger amounts can be separated on preparative columns, or by successive runs with 50 nmol aliquots.

12 Left: elution profile obtained from TSK-G3000 SW column in 6M guanidine- HCl. Right: Calibration curve for MW determinations. K AV 0.333

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15 Separation of proteins on TSK 3000WW column D.C. Harris, Quantitative Analysis, 8th ed., WH Freeman & co., 2010

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17 Most currently evaluated macromolecular contrast agents for magnetic resonance imaging (MRI) are not biodegradable. The goal of this study is to synthesize and characterize poly(l-glutamic acid) (PG) gadolinium chelates as biodegradable blood-pool MRI contrast agents. Two PG chelates of gadolinium diethylenetriaminepentaacetic acid (Gd- DTPA) were synthesized through the use of difunctional and monofunctional DTPA precursors. The conjugates were characterized with regard to molecular weight and molecular weight distribution, gadolinium content, relaxivity, and degradability.

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19 Synthesis of PG-Bz-DTPA-Gd. The number-average molecular weight of Gd 3+ -chelated polymeric conjugate was about as measured by GPC. The compound contained 12.3% (w/w) of gadolinium. Approximately 24% of Glu repeating units contained Gd.

20 GPC profile of PG-Bz-DTPA-Gd revealed by refractive index (- - -) and light scattering ( ). GPC was run on an TSK-G4000PW column eluted with PBS containing 0.1% LiBr at a flow rate of 1.0 ml/min.

21 Analytical Methods. Gel permeation chromatography (GPC) was performed on a Waters (Milford, MA) high-performance liquid chromatography (HPLC) system. A Viscotek E-Zpro triple detector (Viscotek, Houston, TX) was uused to record refractive index, viscosity, and light-scattering signals. The samples were separated using an TSK-G4000PW 4.6 mm 30 cm column (TosoHaas, Montgomeryville, PA) eluted with PBS containing 0.1% LiBr at a flow rate of 1.0 ml/min. Number-average molecular weights of the polymer conjugates were calculated using Viscotek TriSEC GPC software.

22 Biodegradation of Gd-Chelated PG Polymers. Gadolinium chelated PG polymers were dissolved in PBS buffer (ph 5) at a concentration of 5.3 mg/ml. Cathepsin B was added to the solutions to a final concentration of 10 units/ml. The solutions were incubated at 37 C. At predetermined time intervals, aliquots of the polymer solution were removed for GPC analysis.

23 Biodegradation study of PG-Bz-DTPA-Gd

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