[ C O N T R I B U T I O S NO.
81 FROX
R.SABIN LABORATORIES, UNIVERSITY OF
THE D E P A R T M E N T O F BIOPHYSICS,FLORENCE COLORADO MEDICAL CENTER]
Effect of Binding of Ions and Other Small Molecules on Protein Structure. VI. Influence of pH and Chemical Modification on the Electrophoretic Behavior of Proteins in Acidic Medial BY JOHN R. CANNAND ROBERTA. P~IELPS RECEIVED MARCH14, 1959 Electrophoretic experiments on ovalbumin and bovine serum albumin in acetate- and formate-containing media at differcnt pH values support our previous conclusion t h a t the electrophoretic behavior of these proteins in acidic media is determined by interaction of the macromolecular ions with un-ionized buffer acid. These experiments as well as others on chemically modified proteins indicate that macromolecular structural parameters are important in determining the effect of buffer acids on electrophoretic patterns.
Introduction induce a type of non-enantiography typical of that A new effect of acetate (Nahc-HAC), formate actually observed under a variety of conditions (XaF-HF) and other carboxylic acid buffers on of buffer composition, The present communication presents a study oi the electrophoretic patterns of ovalbumin, bovine ?-pseudoglobulin and bovine serum albumin (BSA) the influence of pH and chemical modification on in acidic media has been described previously.2-6 the electrophoretic behavior of ovalbumin, BSA The nature of the electrophoretic patterns of these and ribonuclease (RNAase) in acetate- and forproteins at pH 4 depends upon the concentration mate-containing media. I n addition to supportof buffer in the supporting medium. Increasing ing our previous conclusion concerning the role the concentration of buffer at constant pH and of undissociated buffer acid in determining the ionic strength results in progressive and character- electrophoretic behavior of proteins in acidic media, istic changes in the patterns, notabIy in the ap- the results of these studies indicate that macropearance and growth of fast-moving peaks at the molecular structural parameters are important in expense of slow ones. (See, for example, Figs. 1 determining the effect of these acids on electroand 2 of ref. 3 and Fig. 2 of ref. 5). This behavior phoretic patterns. has been interpreted in terms of complexing of the Experimental protein with undissociated buffer acid with conThe methods used for electrophoretic analysis in the comitant increase in electrophoretic mobility. The Tiselius apparatus have been described in detail previously.2-6 extreniely non-enantiographic nature of the pat- I n all schlieren patterns shown in the figures the apparent terns is a reflection of such interaction and is not mobilities u were p,ositive. The values of 105 X u volt-], are given in the figures. In general, these due t o the relatively high protein concentration sec.-l values cannot be placed in correspondence with mobilities, (1 to 1.37,) per se. since it has been shown that very large changes in conIt has also been shown6 that the various peaks ductance and pH occur in the electrophoresis cell during in the electrophoretic patterns of BSX and oval- passage of the current .6 Crystalline ovalbumin were prepared by the method of Sorensen and H0yrup.' The bumin in Saa4c-HAc at pH 4 correspond neither BSA was Armour's crystallized plasma albumill. The to single stable protein components nor to single RSAase was Pentex's 5 X crystallized material. BSA components involved in a slowly adjusted equi- v a s esterified in acidic methanol according to the method librium. Resolution of the patterns into multiple of Fraenkel-Conrat and Olcott .8 Esterified-BS.4 was by slow addition of acetic anhydride to a solution moving boundaries is intimately related to the acetl-lated of the protein derivative in 0.1 &f XaAc cooled to O0.$ production of conductance and PH gradients during RNAase was oxidized with performic acid according to the electrophoresis. However, it should not be in- inethod used by Harriiigton and Scliellmaii'o to prepare their ferred from this that BSA and ovalbumin do not sample 3. undergo some reaction during electrophoresis. Results and Discussion Rather, it would appear that resolution of the Influence of pH.-A complex dependence of the patterns results from a coupling of such reactions electrophoretic behavior of ovalbumin and BSA with electrophoretic transport of the ions of the supporting medium. An idealized qualitative upon the pH of their acidic solutions was revealed explanation of the patterns has been proposed.6 by preliminary electrophoretic analyses carried This explanation assumes that fast-moving pro- out on protein solutions equilibrated by dialysis tein-acid complexes exist in instantaneous equi- against 0.04 ill- N a F buffers containing varying librium with the undissociated buffer acid as well concentrations of H F . Representative clectroas other constituents of the supporting medium and phoretic patterns of ovalbumin at sex-era1 PH predicts that during electrophoresis the pH in the values are shown in Fig. 1. At PH 4.50the deTiselius cell will change in such a manner as to scending pattern shows a large and sharp slow( I ) S u p p o r t e d in part b y reswrrh g r a n t N o . E-1482 froin t h e Mational I n s t i t u t e of Allergy a n d Infectious Diseases o f the National Institutes of Health, Public Health Service, and in pnrt by t h e Damon K u n q o u Fuud and t h e Arncrican Cancer Sncirty. f%) K. A . Phelps and J. I < . C a n n . THISJ U W R X A I , . IS, :3539 (11)3R). (:i) J I-, I
