ISOELECTRIC-FOCUSING IN A MULTICOMPARTMENT ELECTROLYZER WITH ZWITTERIONIC MEMBRANES, EXEMPLIFIED BY PURIFICATION OF GLUCOAMYLASE

A highly purified preparation of glucoamylase G1 from Aspergillus nige r was found, by isoelectric focusing in immobilized pH gradients, to c ontain a major form with a pI of 3.50 and a number of minor, more acid ic and more basic contaminants. The major isoform was purified to homo geneity by recycling isoelectric focusing in a multicompartment electr olyzer, by confining this form in between two zwitterionic membranes, with pI 3.49 at the anodic side and pl 3.52 at the cathodic side. Reco veries were high (90%) and, notwithstanding the rather low operational pH, the electrosmotic flow was minimal and no protein precipitation o ccurred up to concentrations of 2.5 mg/ml (at the isoelectric point). The forms resolved in an analytical focusing gel were subjected to two types of in situ enzyme detections, by the glucose oxidase peroxidase (GOP) test and by the starch-iodine test. By both criteria all resolv ed zones exhibited enzyme activity, the GOP assay, however, following more closely the Coomassie blue stained protein profile. By computer m odelling, it is shown that it is impossible to obtain linear pH gradie nts at such low pH values (pH 2.5-4.5 intervals) when the mixture has a low buffering power (beta = 2.0 mequiv.l-1 pH-1). When the beta powe r was gradually raised (beta = 4, beta = 6, beta = 8) the pH gradient became progressively linear until, in a recipe with beta = 10 mequiv.l -1 pH-1 full linearity of the pH gradient could be obtained. This is s hown to be due to the substantial buffering power of bulk water in the -pH 2.5-3.5 region.