Separation of Folk Acid Compounds by Gel Chromatography on Sephadex G-15 and G25 *

The separation of folate compounds has been achieved by gel chromatography and the effect of buffer concentration on the elution pattern of folate derivatives was investigated by Sephadex G-15 and G-25 column chromatography. Highly conjugated polyglutamates appear in the effluent considerably sooner when eluted with water than would be expected from their molecular weights. Monoglutamates, except IO-formyltetrahydropteroylglutamic acid, are retarded on G-15 and G-25 in phosphate buffer (pH 7.0) solvent system; the situation becomes more apparent as the buffer concentration is increased. Chromatography on Sephadex G-25 with 0.1 M phosphate buffer, pH 7.0, containing 0.2 M E-mercaptoethanol gives a satisfactory separation of pteroylpolyglutamates containing 2 to 7 glutamic acid residues.

The separation of folate compounds has been achieved by gel chromatography and the effect of buffer concentration on the elution pattern of folate derivatives was investigated by Sephadex G-15 and G-25 column chromatography.
Highly conjugated polyglutamates appear in the effluent considerably sooner when eluted with water than would be expected from their molecular weights.
Monoglutamates, except IO-formyltetrahydropteroylglutamic acid, are retarded on G-15 and G-25 in phosphate buffer (pH 7.0) solvent system; the situation becomes more apparent as the buffer concentration is increased. Chromatography on Sephadex G-25 with 0.1 M phosphate buffer, pH 7.0, containing 0.2 M E-mercaptoethanol gives a satisfactory separation of pteroylpolyglutamates containing 2 to 7 glutamic acid residues.
Polyglut:~ruatc forms of folic acid are known to be the major forms of rl:Ltur:tl folatcs in animal tissues (1,2), plants (3), yeast (4), and bacteria (5). The identification and separation of these compour~ds was based mainly on ion exchange column chromatography together with microbiological assays (1,3). However, this method has not proved suitable for the determination of the Ilumber of glutamic acid residues in polyglutamate forms of folic acid.
Gel chromatography, commonly used for the separation of molccul~ of different sizes, has been neglected for the separation of 1~olyglutamnt.e forms of folic acid. Recently, Whitehead used Ilio-Gel chromatography for the separation of pteroylmottoglutamates from di-and triglutamates in liver (6). Another approach to determine the chain length of polyglutamates involves the degradation of natural folates to their corresponding p-amino-benzoyll~olyglutan~~l derivatives (7). In our study, we developed a method to separate natural folates containing 1 to 7 glutamic acid residues on Sephadex G-15 aud G-25 coIumns and examined the influence of various buffer concentrations on the elution volume of these compounds. * This work was supported by United Sta.tes Public Health Service (:rnnt AM-OS171 from the Nationat Institutes of Heaith. MATERIALS AND METHODS p-Aminobenzoic acid and folic acid were purchased from Sigma. Tritiated folic acid was obtained from Amersham-Searle and 5-CHO-H4PteGlu1 from Lederle Laboratories. HJ'teGlu, was prepared by the reduction of folic acid with hydrogen with platinum as the catalyst (8). lo-CHO-HIPteGlu, was prepared from 5-CHO-H4PteGlu, (9) or from PteGlu, (10). 5-CH3-HZteGlu, was synthesized by reducing 5, lO=CH-HIPteGlu, with sodium borohydride (11). Pteroic acid was obtained from Lederle.
Pteroic acid was also prepared by incubation of folic acid with a strain of Pseudo?nonas (12). Radioactive polygluta- mates of pteroic acid, PteGlu,-2-iU-1"C]Glu-Glu, and the respective nonradioactive compounds were prepared according to the method of Laugh and coworkers (13,14) and purified by chromatography on QAE-Sephadex A-25 (15). Sephadex G-15 and G-25 (fine) were swollen for I hour in a boiling water bath, equilibrated with the respective buffer, and stored at 4" until used. A glass column (0.75 x 200 cm) was packed by gravity to a height of 200 cm and washed with 500 ml of the buffer to be used as eluant.

RESULTS AIiD DISCUSSION
The chromatographic behavior of the compound studied is expressed by its K,, value (16), which is calculated as K,, = V, -Vo/Ft -B0 where V0 is the exclusion volume, V, the elution volume, and V, the volume of the gel bed. VO was determined as the elution volume of blue dextran and V, as the effluent volume at which the concentration of the elutcd substance is maximum. Table I shows the results of chromatography of pteroylmonoglutamates on Sephadex G-15 and G-25. No difference in the Ii,,, values of different derivatives of folic acid was obtained when distilled water was used as eluant.
There are in essence three different types of interactions in gel chromatography: first, interactions between ion-charged regions in the gel; second, Van der Waals interactions; third, adsorption of t.he substance onto the structure which forms the gel matrix. The interaction betweerr charged groups can easily be eliminated by increasing the salt concentration of the buffer. This is not the case with folic acid, since an increase in the buffer strength gives stronger interaction between the solutes and the gel (Table  I). Although it is reported that adsorption is generally observed in the case of aromatic or hetjerocyclic compounds, how this adsorption occurs is not completely understood.
Substitution with the formyl group in position 10 and to a lesser extent in position 5 of the folic acid molecule interferes with adsorption on the gel matrix.
The glutamic acid moiety of folic acid also diminishes this adsorption.
Whereas pteroic acid is retarded considerably even in a distilled water solvent system, the elution volume of polyglutamate forms of folio: acid is much less affected by an increase in the buffer strengt,h, as shown in Table II. Polyglutamates appear in the effluent considerably sooner than would be expected from their molecular weight in pure aqueous solvent system. This phenomenon remains largely unexplained. However, it seems to be related to the negative charges of the gel (0.1 to 0.2 meq of the carboxyl group per g dry Sephadex), which results in the exclusion of negatively charged molecules (17). The presence of electrolytes somewhat normalizes the elution pattern. K,, values of polyglutamates, too, are increased in higher concentrations of buffer eluant; however, to a much lesser extcrit with increasing number of glutamic acid residues. The behavior of reduced derivatives of polyglutamates is slightly different from that of oxidized compounds (Table II). 10.CHO-HaPteGlus is eluted sooner than the unsubstituted compound. K,, is decreased by 0.07 to 0.09 and the K,, of 5-CHs-PteGluS is decreased by 0.02 to 0.03 in comparison with PteGlu5.
The elution positions of the folate compounds on a Sephadex G-25 column in 0.1 M phosphate buffer (pH 7.0) are shown in Fig. 1. Figs. 2 and 3 show t,wo examples of the application of Sephadex column chromatography on the separation of naturally occurring folate compounds.
According to Fig. 2, folate derivatives in rat kidney exist mainly as reduced pentaglutamates, and