Subtilisin enzymes: A note on time‐resolved fluorescence and circular dichroism properties

Abstract

This note briefly corrects previous information about the time-resolved fluorescence properties of preparations of subtilisin Carlsberg and subtilisin BPN'. We confirm the observation of segmental motion of the single tryptophan in subtilisin Carlsberg by analysis of the time-resolved fluorescence anisotropy, and present circular dichroism and spectroscopic data on the two proteins. Near-UV properties clearly differentiate between the two proteins. Far-UV circular dichroism confirms that the two subtilisins have closely similar secondary structure in solution; the multi-component analysis is consistent with the established X-ray conformations, but the quantitative agreement is still somewhat imperfect.

Role of surfactant on the proteolysis of aqueous bovine serum albumin

“…These results indicate that the enzyme does not degrade in the presence of SDS, DTAB, or itself over a 10 h period. We conclude that subtilisin Carlsberg does not autolyze during the time of our experiments with or without surfactant, in agreement with Bayley et al (1989) who found negligible subtilisin Carlsberg autolysis at concentrations <10 mM in neutral pH. Figure 5 shows that when the ionic surfactants are added to FBSA solution, fluorescence intensity increases until near the CMC.…”

“…Likewise, partial unfolding of enzymes due to surfactant generally results in loss of enzymatic activity (Cruzten and Douglas, 1999;Stoner et al, 2004). However, sodium alkyl benzene sulfonate (Nacconol 90G), and sodium lauryl sulphate (Steol CS-370) increase subtilisin Carlsberg autolysis rate in a concentrated heavyduty-liquid-detergent formulation (Stoner et al, 2004), whereas sodium dodecyl sulphate (SDS) has no effect for enzyme concentrations below 10 mM (Bayley et al, 1989).…”

Progressive Rearrangement of Subtilisin Carlsberg into Orderly and Inflexible Conformation with Ca2+ Binding

“…This indicates that the fractions calculated from CD spectra have large errors inherently. Thus we estimated just the approximate fractional changes of the constituting secondary structures with Ca 2ϩ removal, considering the secondary structure fractions of NsC by Bayley et al (1989) and the basis spectra and estimation method of secondary structures by Saxena and Wetlaufer (1971). Despite large uncertainties, we are showing experimentally for the first time the progressive structural changes of sC with Ca 2ϩ binding.…”

“…The determination of individual secondary structure fractions by analyzing CD spectra has been widely used (Venyaminov and Yang, 1996). Although the fractions of ␣-helix, ␤-sheet, turn, and random coil in NsC are reported as 0.31, 0.1, 0.22, and 0.37, respectively, from the x-ray structure (Chang et al, 1978), they are found to be 0.22, 0.47, 0, and 0.31, respectively, from the CD spectrum (Bayley et al, 1989). This indicates that the fractions calculated from CD spectra have large errors inherently.…”

“…The fast depolarization time depends on the monitored wavelength strongly, whereas the slow one does not at all. The slow one of 12 ns is ascribed to the overall rotation time of the entire polypeptide because the global rotation time measured from the Trp fluorescence depolarization kinetics of sC is reported to be 11.3 ns (Bayley et al, 1989) and the time is calculated using the Stokes-Einstein relation as 12 ns for the 30 kDa protein of sC (Cantor and Schimmel, 1980). Then the fast component arises from the internal rotation of Tyr and Trp fluorophores with respect to the entire protein motion.…”