Evidence that the two free sulfhydryl groups of plasma fibronectin are in different local environments. Saturation-recovery electron spin resonance study.
Human plasma fibronectin is a dimer consisting of two subunits; each contains two cryptic thiol groups that were selectively labeled with an 15N,2H-maleimide spin label. Previous studies using conventional X-band electron spin resonance (ESR) methods showed that the spectrum of the labeled protein displays a single strongly immobilized component with an effective rotational correlation time of approximately 17 ns, suggesting that the physical environments of the two labeled sites per chain are indistinguishable. Here we have used saturation-recovery ESR to measure directly electron spin-lattice relaxation time (T1) of the labeled protein in solution at 27 degrees C. Interestingly, the time evolution of the signal was found to be biphasic, which was deconvoluted into two T1 values of 1.37 and 4.53 microseconds. Thus, the two spin-labeled sulfhydryl sites of plasma fibronectin (Fn), being similar in rates of rotational diffusion, differ by a factor of 3.2 in T1. Parallel experiments using various fibronectin fragments showed that the 1.37-microseconds component is associated with the label attached onto the thiol located in between the DNA-binding and the cell-binding domains, and the 4.53-microseconds component is associated with the label attached onto the thiol located within the carboxyl-terminal fibrin-binding domain. The data suggest that the saturation-recovery ESR is a useful method for differentiating multiple spin-labeled sites on macromolecules in which the labels undergo similar rates of rotational motion.
Lai
C S
CS
National Biomedical Electron Spin Resonance Center, Department of Radiology, Medical College of Wisconsin, Milwaukee 53226.
Narasimhan
C
C
Yin
J J
JJ
D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005292:Ferricyanides; D005353:Fibronectins / Q000097:blood*; D006801:Humans; D046911:Macromolecular Substances; D011487:Protein Conformation; D013113:Spin Labels; D013438:Sulfhydryl Compounds
D005292:Ferricyanides; D005353:Fibronectins; D046911:Macromolecular Substances; D013113:Spin Labels; D013438:Sulfhydryl Compounds; C028033:potassium ferricyanide