Focusing of alkaline proteases (subtilisins) in pH 10-12 immobilized gradients.
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.
Bossi
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Chair of Biochemistry, Faculty of Science, University of Calábria, Italy.
Righetti
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PG
Vecchio
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Severinsen
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D000595:Amino Acid Sequence; D004800:Enzymes, Immobilized; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013381:Subtilisins / Q000737:chemistry*
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; C093184:Durazym; D012697:Serine Endopeptidases; D013381:Subtilisins; C032923:microbial serine proteinases