Abstract
Sodium dodecyl sulfate (SDS) dissociates the multimeric protein, carmin, into its monomers (2 S) at low concentration. Dissociation begins at and above 1.6 mM SDS concentration and reaches 50% at 5 mM SDS concentration. Denaturation occurs above 5 mM SDS concentration. The dissociation step involves binding of 540 +/- 50 mol of SDS/mol of protein with an association constant, K alpha of 6.90 +/- 0.35 x 10(2) M-1. The interaction reflects a delta G(0) = -4.0 +/- 0.1 kcal mol-1. In the denaturation step, the K alpha has the same value, and the gamma value is nearly 2-fold higher. Dissociation of carmin thus begins only above a binding of 0.60 g of SDS/g of protein. Analysis of the binding data at 37 degrees C indicates a maximum of 1030 +/- 90 mol of SDS bound/mol of protein, which is equivalent to 1.14 +/- 0.10 g of SDS/g of protein. Upon denaturation, the alpha-helix content of the protein increases from 4 to 15%. Kinetically, the denaturation process consists of a two-step process (a fast and a slow step). The first order rate constants for these steps are 89.6 +/- 8.1 and 15.8 +/- 1.5 min-1, respectively, at 6.3 mM SDS concentration. The processes of dissociation and denaturation occur sequentially. Dissociation of the protein is reversible, whereas the process of denaturation is only partially reversible as reflected by sedimentation velocity and conformational analysis. These data are taken as a model for general understanding of the dissociation and/or denaturation processes, which could be either sequential or simultaneous in multimeric proteins.
Highlights
Sodium dodecylsulfate (SDS) dissociates the multi- and kinetics of the process, to elucidate the mechanism of meric protein, carmin,into its monomers (2 S ) at low dissociation, are very limited (Jones et al, 1982a, 1982b)
Concentration.Dissociation begins at andabove 1.6 Carmin, the high molecular weight protein from safflower mM SDS concentration and reache5s 0%at 5 mM SDS seed (Carthamus tinctorius L.), is characterized and isknown concentration
The the aim of this study to find out if the processes of dissociation denaturation processconsists of a two-step process
Summary
At 50.6 "C, the maximum number of binding sites was found to be 440 f 40 with a AG" value of -3.10 f 0.15 kcal mol" This is equivalent to 0.49 f 0.04 g of SDS bound/ g of protein. Carmin has a value of 12 ml/g to 1 X lo-’ M SDS), theprotein obtained by removal of SDS at pH 2.0 (Sudhindra Rao and Prakash, 1989), 32 ml/g in 6 from the denatured molecule to generate dissociated state, M urea (Prakash, 1986), and 5.5 ml/g at pH 12.5 (Rajendran and no SDS state all have their emission maximum at 337 and Prakash, 1988). Carmin moval of SDS, the helical content decreases by 3% for disso- under different denaturing conditions shows similar red ciated to no SDS state and from 15 to 10% from denatured shift in the fluorescence emission maximum: 336 nm at pH to no SDS state (Fig. 6). 12.3 (Rajendran and Prakash, 1988), 337 nm at pH 2.5 (Sudhindra Rao and Prakash, 1989), and 337 nm in 6 M urea
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