Discussion on the paper of Professor Sorm 



B. KEIL 



Allow me to supplement Professor Sorm's lecture by a few remarks concern- 

 ing methods and techniques. In the work discussed by Professor Sorm we 

 have been using a number of procedures worked out in our laboratories, 

 in addition to the well-known and well-tried methods of separation and 

 identification. For following the course of fractionation on Dowex columns 

 or by multi-compartment electrophoresis we use paper electrophoretic ana- 

 lysis of samples in place of the usual ninhydrin colorimetry. This method 

 gives rapid approximate information not only of the amount, but also 

 of the nature of the material in the samples. The arrangement used for 

 this purpose is very simple.^ A sheet of Whatman paper is loosely 

 stretched vertically between two troughs containing pyridine acetate buffer 

 of a low and defined conductivity. Voltages as high as 1500 V, corre- 

 sponding to a potential gradient of about 40 V/cm., can be appUed without 

 cooling (Fig. 1). 



The analytical separation is very uniform ; the time of each run is 1 -0 to 

 1-5 hours. Up to 200 mg. of a peptide mixture can be resolved on a single 

 sheet of Whatman No. 3 paper. Sanger has used a four-compartment elec- 

 trophoretic apparatus for the primary fractionation of acid hydrolysates of 

 insulin. We have found a five-compartment apparatus most convenient, with 

 formic acid, acetic acid and ammonia as the electrolytes.^ The omission of 

 the sulphuric acid and the introduction of the fifth compartment removes 

 the danger of loss or contamination of the material at the electrodes. The 

 separation of partial hydrolysates into basic, acidic and neutral peptide 

 fractions is clear-cut, as may be seen from the paper electrophoretic check 

 of the individual fractions. 



An extension of the membrane system of electrophoresis led to the de- 

 velopment in our laboratories of a multi-compartment apparatus for the 

 fractionation of peptides and proteins.^ In a set-up of one hundred com- 

 partments, with water-cooling and pyridine acetate buffer, and working at 

 5000 V, a progressive fractionation of peptide mixtures can be achieved 

 practically without losses. The fractions are withdrawn from the individual 

 compartments by pipetting, and directly lyophilized. A similar arrangement 

 of twenty compartments with paper membranes has been used for the pre- 

 parative fractionation of proteins and of mixtures of nucleic acids, poly- 

 saccharides and proteins (Fig. 2). 



