Low molecular weight markers

Protein concentration was determined by the method of Bradford [14] (link), using a Dc protein assay kit purchased from Bio-Rad Laboratories (Inc., Hercules, CA, USA) with bovine serum albumin as a reference. Analytical soduim dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed following the method of Laemmli [15] (link). Protein bands were visualized with Coomassie Brilliant Blue R-250 staining. Casein-zymography analysis was performed as previously described elsewhere [9] (link). Low molecular weight markers from Amersham Biosciences were used as protein marker standards. The molecular masses of the selected purified SAPB-L31I, SAPB-T33S, and SAPB-N99Y enzymes were analyzed in the linear mode by MALDI-TOF/MS using a Voyager DE-RP instrument (Applied Biosystems/PerSeptive Biosystems, Inc., Framingham, MA, USA). Bands of purified enzymes (SAPB-L31I, SAPB-T33S, and SAPB-N99Y) were separated on SDS gels and transferred to a ProBlott membrane (Applied Biosystems, Foster City, CA, USA). N-terminal sequence analyses were performed by automated Edman’s degradation using an Applied Biosystem Model 473A gas-phase sequencer.