Figure 1.
X-ray diffraction pattern of precipitate recovered from the solutions as described in the text.
Red: control experiments, no protein; blue: [nOPN] = 1 µg/ml. The control data have been offset vertically for clarity. The positions of the peaks in 2θ using Cobalt Kα radiation in the reference pattern for hydroxyapatite are shown at the bottom of the figure (ICDD card #01-073-0293).
Figure 2.
A typical intensity autocorrelation function, , obtained from a control experiment with no protein.
The heavy dashed curve is a fit to the cumulant expansion given in Eq. (3), performed on data with s and
to minimize the influence of noise at short and long lag times. The dotted curve is an extrapolation of the fit over the rest of the measured τ range.
Figure 3.
Precipitation of calcium phosphate in the absence of protein.
(A) Scattered photon count rate and (b) hydrodynamic radius
obtained from an experiment with no added protein. The error bars in (A) indicate typical values of the standard deviation of the count rate. In (B) they indicate the standard deviation of the distribution of particle sizes determined from fits of Eq. (3) to the intensity autocorrelation function. Both the count rate and
increase over the course of the experiment as the particles of calcium phosphate precipitate grow.
Figure 4.
Precipitation of calcium phosphate in the presence of nOPN.
(A) Mean scattered intensity and (B) hydrodynamic radius plotted as functions of time for a range of concentrations of native osteopontin, nOPN. (C) shows as a function of
. black circles: control, [nOPN] = 0 µg/ml; red circles: 1 µg/ml (0.03 µM); crosses: 1.4 µg/ml (0.05 µM); squares: 2 µg/ml (0.06 µM); upward-pointing triangles: 3 µg/ml (0.09 µM); downward-pointing triangles: 4 µg/ml (0.12 µM). Error bars in (A) indicate typical standard deviations in the photon count rate for the control run and for [nOPN] = 1.0 µg/ml. In (B), they show the standard deviation
of the distribution of particle sizes for the control run. The values of
in the protein runs are similar.
Figure 5.
Delay time for the onset of crystal growth as a function of protein concentration.
Data are plotted for both nOPN and p-rOPN. Concentrations lower than those plotted had no measurable delay. The dotted lines are linear fits to the data for each protein.
Figure 6.
Growth rate of the precipitating partcles at = 200 nm plotted as a function of protein concentration.
Figure 7.
Precipitation of calcium phosphate in the presence of rOPN.
(A) Mean scattered intensity and (B) hydrodynamic radius plotted as functions of time for a range of concentrations of recombinant osteopontin, rOPN. (C) shows
as a function of
. black circles: control, [rOPN] = 0 µg/ml; red circles: 0.5 µg/ml (0.01 µM); crosses: 1 µg/ml (0.03 µM); squares: 2 µg/ml (0.06 µM); upward-pointing triangles: 3 µg/ml (0.08 µM); downward-pointing triangles: 6 µg/ml (0.17 µM). Error bars are analogous to those in Figure 4.
Figure 8.
Precipitation of calcium phosphate in the presence of p-rOPN.
(A) Mean scattered intensity and (B) hydrodynamic radius plotted as functions of time for a range of concentrations of phosphorylated recombinant osteopontin, p-rOPN. (C) shows
as a function of
. black circles: control, [p-rOPN] = 0 µg/ml; red circles: 1.5 µg/ml; crosses: 3.1 µg/ml; squares: 6 µg/ml; upward-pointing triangles: 8 µg/ml. Error bars are analogous to those in Figure 4.
Figure 9.
Constant-composition measurements of seeded HA crystal growth.
The total volume of titrant added as a function of time in the constant-composition seeded growth experiments in the presence of (A) nOPN and (B) rOPN. The different curves correspond to different concentrations of protein. In (A), the [nOPN] is, from top to bottom, 0, 2.7, 8.0, and 15.9 µg/ml (0, 0.07, 0.21, and 0.42 µM). In (B), [rOPN] is, from top to bottom, 0, 5.0, and 15.0 µg/ml (0, 0.14, and 0.41 µM).
Figure 10.
HA growth rate from the constant-composition experiments.
The growth rate of the HA seed crystals relative to that in the absence of protein is plotted as a function of protein concentration for the constant-composition experiments. The dashed lines are straight lines fit to the data.