Focusing of alkaline proteases (subtilisins) in pH 10–12 immobilized gradients

Abstract

Isoelectric focusing in very alkaline immobilized pH gradients (IPG) was adopted for checking the purity and assessing the pI value of two strongly alkaline proteases: Savinase and Durazym. The first enzyme (known to be the most alkaline) contains 5 Asp, 5 Glu, 7 His, 7 Tyr, 5 Lys, no Cys and 8 Arg residues and should have a theoretical pI of 9.7. Yet, when focused in a pH 9-11 IPG interval, it was lost in the cathodic compartment. After repeated attempts at creating even more alkaline pH intervals, a pH 10-12 IPG range was finally optimized and proved successful in focusing both enzymes midway between the two electrodic compartments. The pI of Savinase was measured as 11.15 +/- 0.15; that of Durazym as 10.95 +/- 0.20 and that of the pI marker cytochrome c as 10.6 +/- 0.17. Both enzymes (and a number of minor components in each preparation) were proven to be active by an in situ zymogram consisting of a casein/agar overlay. The discrepancy between theoretical and experimental pI values could not be fully reconciled: when correcting for pK values of amino acids in proteins at 10 degrees C, instead of the tabulated values at 25 degrees C, the pI should increase to a value of 10. Differential UV spectra showed that ca. 1/2 Tyr are buried in the protein interior and are thus unable to contribute to surface charge. This further increases the pI value by 0.3 pH units to a value of 10.3, still quite removed from the experimentally assessed pI value (in the gel, at 10 degrees C) of 11.15.

Alkaline proteins ofBacillus subtilis: First steps towards a two-dimensional alkaline master gel

“…At very high pH the polyacrylamide matrix is positively charged and a strong electroendosmotic flow (EOF) to the anode is created, which is opposite to the direction of the EOF typically observed when the gel matrix is negatively charged at lower pH. This results in a strong water transport from the cathode to the anode and a change of charge of the IPG matrix, disturbing the steady-state point of the proteins [8,9,11].…”

Alkaline proteins of Bacillus subtilis: First steps towards a two-dimensional alkaline master gel

“…The development of acrylamido buffer systems with a pK value >13 allowed us to create narrow IPGs at the extreme alkaline range, which should provide reproducible and highly resolved steady-state 2-D patterns of alkaline proteins. This results in a strong water transport from the cathode to the anode and a change of charge of the IPG matrix, disturbing the steady-state point of the proteins [8,9,11]. At very high pH the polyacrylamide matrix is positively charged and a strong electroendosmotic flow (EOF) to the anode is created, which is opposite to the direction of the EOF typically observed when the gel matrix is negatively charged at lower pH.…”

Very alkaline immobilized pH gradients for two‐dimensional electrophoresis of ribosomal and nuclear proteins

“…a more acidic (pH [3][4] or more alkaline (pH 10-11) IPG interval, one is faced with severe problems as the "buffering power" of water becomes nonnegligible, while outside these limits it rises so sharply as to nullify any attempt at using IPGs in these regions [17]. More recently, by using Immobiline pK > 13, a nonlinear IPG spanning a 10-12 interval was generated and optimized for IEF [18,191. First results of 2-D electrophoresis using IPG 10-12 were obtained for the separation of commercial histone preparations [20].…”