Photon and fluorescence correlation spectroscopy and light scattering of eye-lens proteins at moderate concentrations

Abstract

The bovine eye-lens protein, alpha L-crystallin, has been studied with photon correlation spectroscopy to obtain the mutual diffusion coefficient, Dm, with fluorescence correlation spectroscopy to determine the tracer diffusion coefficient, DT, and with light scattering to get the isothermal osmotic compressibility (delta pi/delta c) P,T. The concentration dependence of Dm, DT, and (delta pi/delta c) P,T up to a volume fraction phi of the protein of 2.5 x 10(-2) has been interpreted on the basis of four different interaction potentials: (a) an extended hard-sphere potential; (b) a shielded Coulomb potential; (c) a shielded Coulomb interaction where the effect of counterions is included; (d) a simple mixed potential. The three parameters Dm, DT, and (delta pi/delta c) P,T have also been combined in the generalized Stokes-Einstein equation, Dm = [(delta pi/delta c)P,T . (1--phi) . (DT)]/(kappa B . T). Our results indicate that, in the case that photon correlation spectroscopy gives the mutual diffusion coefficient Dm, the applicability of the Stokes-Einstein equation can be questioned; or that, when one assumes the Stokes-Einstein equation to be valid, there is significant discrepancy between the result of photon correlation spectroscopy and Dm.

Photon correlation spectroscopy and light scattering of eye lens proteins at high concentrations

“…Initial Decay: Concentration Dependence. At low and moderate volume fractions X < 0.035, the correla-tion function is a monoexponential function and as a consequence the Q factor is small (Andries et al, 1983). However, from 4 = 0.035 on, important changes are noted in the diffusion behavior of the aL-crystalline solutions.…”

“…The preparation of aL-crystallins has been described elsewhere (Andries et al, 1983). For the higher concentration studies, the material from two zonal centrifugations had to be collected and concentrated to a final volume of I ml.…”

“…Extra care was taken to avoid the accumulation of dust by the concentrating procedure; we have added an extra centrifugation step between the concentrating step on an Amicon concentration cell and the final concentrating on a Minicon B sample concentrator (Amicon Corp., Scientific Sys. Div., Danvers, MA), to the procedures already described before (Andries et al, 1983).…”

“…The apparatus and the precautions, which have been taken during photon correlation spectroscopy and light-scattering measurements, have been described (Andries et al, 1983). Because the correlation function at higher protein concentration is no longer single-exponential, the Malvern (type 7023; Malvern Instruments, Malvern, Worchestershire, England) has been extended to 96 channels.…”

“…The use of light scattering and photon correlation spectroscopy under identical conditions gives similar and complementary information. Photon and fluorescence correlation spectroscopy and light scattering of aL-crystallin up to a volume fraction of 2.5 x 10-2 have taught us that the interaction potential between the proteins at moderate ionic strength (I = 0.08) and higher ionic strength (I = 0.32) can be described by an extended hard-sphere potential or a shielded coulomb potential (Andries et al, 1983).…”