OSTEOCALCIN ADSORPTION ONTO THE SILICA AND HAP
OSTEOCALCIN ADSORPTION ONTO THE SILICA AND HAP SURFACES L. A. Scudeller1,2*; A. M. P. Silva1; A. M. Rossi1; D. G. Castner 2 1 Centro Brasileiro de Pesquisas Físicas, Rua Dr. Xavier Sigaud, 150, Urca, 22290-180, Rio de Janeiro, RJ, Brazil. 2 NESAC/BIO, 220 Molecular Engineering & Sciences Building, University of Washington, 98195-1653, Seattle, WA, USA.
This study used XPS and ToF-SIMS to investigate the adsorption of osteocalcin (OC) and decarboxylated OC (dOC) onto various calcium phosphate and silica surfaces. The largest differences were observed between OC and dOC adsorbed onto the silica and HAp surfaces. Similar amounts of both proteins were adsorbed onto the silica surface while higher amounts were adsorbed on the HAp surface. The ToF-SIMS data showed the intensity of the Cys amino acid fragment, normalized to intensity of all amino acid fragments, was significantly higher (~x10) when the proteins were adsorbed onto silica. No significant differences were detected between OC and dOC adsorbed onto the silica surface, but small differences were observed between OC and dOC adsorbed onto the different HAp surfaces.
Keywords: Osteocalcin, Hydroxyapatite, ToF-SIMS, XPS, Surface.
Introduction Osteocalcin (OC) is the most abundant, non-collagenous protein in bone and accounts for almost 2% of total protein in the human body [1]. OC plays a role in the body's metabolic regulation and bone building, as well as being used as a biochemical marker for bone formation. However, its precise function is not known, OC is known to bind strongly to hydroxyapatite (HAp). This strong binding is likely the result of the γcarboxylated glutamic acid residues (Gla) in OC interacting with Ca2+ ions on the HAp surface [2]. Experimental part We put 500 µL of protein solution directly on silicon wafer and let at room temperature during 3 h to complete the adsorption. For HAp powder, we mixed 0.1 mg of HAp and 100 µL of protein solution letting the solution mixing during 3 h in a vortex mixer at room temperature. We made 3 triplicates for each sample and each protein. After rinse, we dropped 20 µL of HAp with protein on Si wafer and let dry overnight. The Si wafer with protein and with HAp+protein were characterized by X-Ray Photoelectric Spectroscopy - XPS and Time-of-Flight Secondary Ion Mass Spectroscopy - ToF-SIMS to compare the adsorption of both protein on both surfaces. Results and discussion While similar amounts of OC and dOC were adsorbed onto the silica surface, higher amounts were adsorbed on the HAp surface (~5 atomic % N for dOC and ~8 atomic % N for OC), Fig. 1a. Furthermore, the intensity of the Cys amino acid fragment, normalized to intensity of all amino acid fragments, was significantly higher (~x10) when the proteins were adsorbed onto silica, Fig. 1b. Since in the native OC structure the cysteines are buried in the center of the 3 α-helices [3], this indicates both OC and
dOC are more denatured on the silica surface. Although no significant differences were detected between OC and dOC adsorbed onto Si, small differences were observed for the absorption onto the HAp surface (data not shown). XPS showed similar amounts of OC and dOC were absorbed onto amorphous HAp (HAp5), crystalline HAp (HAp90) and octacalcium phosphate (OCP), Fig. 2a, but ToF-SIMS detected some small differences in the amino acid fragment intensities between adsorbed OC and dOC, Fig. 2b.
Fig. 1 – Results for absorption of dOC and OC onto silica and HAp surfaces (a) XPS showing the % atomic N (b) Cys amino acid normalized by the sum of all amino acids.
Fig. 2 –
Absorption on amorphous
onclusions (HAp5), crystalline (HAp90) and OCP
(a) XPS showing no difference of N amount on different CaP (b) ToF-SIMS showing the separation between OC and dOC.
Conclusions The largest differences were observed between OC and dOC adsorbed onto the silica and HAp surfaces. We could see that in absence of Ca (Si surface), both protein (OC and dOC) are denatured, but on HAp surfaces they are not. We could see a difference between OC and dOC, with a big absorption for OC and a small Cys signal indicating that the α3 helices is on top of α1 and α2 helices [3]. The crystallinity and the composition showed not to make difference in the absorption, since there is no difference on XPS and SIMS data between the different surfaces. The difference is again only between dOC and OC.
References [1] Hauschka P. V., et. al., “Osteocalcin and Matrix Gla Protein: Vitamin K-Dependent Proteins in Bone”, Physicol. Rev. (1989) 69:990-1047 [2] Hauschka P. V. and Carr S. A., “Calcium-dependent α-helical structure in osteocalcin”, Biochemistry (1982) 21:2538-2547. [3] Hoang Q. Q., Sicheri F., Howard A. J., Yang D. S. C., “Bone recognition mechanism of porcine osteocalcin from crystal structure”, Nature (2003) 425:977-980. Acknowledgement: FAPERJ, CNPq
This study used XPS and ToF-SIMS to investigate the adsorption of osteocalcin (OC) and decarboxylated OC (dOC) onto various calcium phosphate and silica surfaces. The largest differences were observed between OC and dOC adsorbed onto the silica and HAp surfaces. Similar amounts of both proteins were adsorbed onto the silica surface while higher amounts were adsorbed on the HAp surface. The ToF-SIMS data showed the intensity of the Cys amino acid fragment, normalized to intensity of all amino acid fragments, was significantly higher (~x10) when the proteins were adsorbed onto silica. No significant differences were detected between OC and dOC adsorbed onto the silica surface, but small differences were observed between OC and dOC adsorbed onto the different HAp surfaces.
Keywords: Osteocalcin, Hydroxyapatite, ToF-SIMS, XPS, Surface.
Introduction Osteocalcin (OC) is the most abundant, non-collagenous protein in bone and accounts for almost 2% of total protein in the human body [1]. OC plays a role in the body's metabolic regulation and bone building, as well as being used as a biochemical marker for bone formation. However, its precise function is not known, OC is known to bind strongly to hydroxyapatite (HAp). This strong binding is likely the result of the γcarboxylated glutamic acid residues (Gla) in OC interacting with Ca2+ ions on the HAp surface [2]. Experimental part We put 500 µL of protein solution directly on silicon wafer and let at room temperature during 3 h to complete the adsorption. For HAp powder, we mixed 0.1 mg of HAp and 100 µL of protein solution letting the solution mixing during 3 h in a vortex mixer at room temperature. We made 3 triplicates for each sample and each protein. After rinse, we dropped 20 µL of HAp with protein on Si wafer and let dry overnight. The Si wafer with protein and with HAp+protein were characterized by X-Ray Photoelectric Spectroscopy - XPS and Time-of-Flight Secondary Ion Mass Spectroscopy - ToF-SIMS to compare the adsorption of both protein on both surfaces. Results and discussion While similar amounts of OC and dOC were adsorbed onto the silica surface, higher amounts were adsorbed on the HAp surface (~5 atomic % N for dOC and ~8 atomic % N for OC), Fig. 1a. Furthermore, the intensity of the Cys amino acid fragment, normalized to intensity of all amino acid fragments, was significantly higher (~x10) when the proteins were adsorbed onto silica, Fig. 1b. Since in the native OC structure the cysteines are buried in the center of the 3 α-helices [3], this indicates both OC and
dOC are more denatured on the silica surface. Although no significant differences were detected between OC and dOC adsorbed onto Si, small differences were observed for the absorption onto the HAp surface (data not shown). XPS showed similar amounts of OC and dOC were absorbed onto amorphous HAp (HAp5), crystalline HAp (HAp90) and octacalcium phosphate (OCP), Fig. 2a, but ToF-SIMS detected some small differences in the amino acid fragment intensities between adsorbed OC and dOC, Fig. 2b.
Fig. 1 – Results for absorption of dOC and OC onto silica and HAp surfaces (a) XPS showing the % atomic N (b) Cys amino acid normalized by the sum of all amino acids.
Fig. 2 –
Absorption on amorphous
onclusions (HAp5), crystalline (HAp90) and OCP
(a) XPS showing no difference of N amount on different CaP (b) ToF-SIMS showing the separation between OC and dOC.
Conclusions The largest differences were observed between OC and dOC adsorbed onto the silica and HAp surfaces. We could see that in absence of Ca (Si surface), both protein (OC and dOC) are denatured, but on HAp surfaces they are not. We could see a difference between OC and dOC, with a big absorption for OC and a small Cys signal indicating that the α3 helices is on top of α1 and α2 helices [3]. The crystallinity and the composition showed not to make difference in the absorption, since there is no difference on XPS and SIMS data between the different surfaces. The difference is again only between dOC and OC.
References [1] Hauschka P. V., et. al., “Osteocalcin and Matrix Gla Protein: Vitamin K-Dependent Proteins in Bone”, Physicol. Rev. (1989) 69:990-1047 [2] Hauschka P. V. and Carr S. A., “Calcium-dependent α-helical structure in osteocalcin”, Biochemistry (1982) 21:2538-2547. [3] Hoang Q. Q., Sicheri F., Howard A. J., Yang D. S. C., “Bone recognition mechanism of porcine osteocalcin from crystal structure”, Nature (2003) 425:977-980. Acknowledgement: FAPERJ, CNPq
