*3.4. Protein Adsorption on Peptide–HAp*

Each protein (i.e., Cyt c, MGB, BSA, LSZ, OVT, and TF) was dissolved in 10 mM phosphate buffer (pH 7.0), and the protein solution was prepared at a concentration of 250 μg mL<sup>−</sup>1. Peptide–HAp (1.5 mg) was mixed with a 1 mL protein solution, followed by stirring overnight at 20 ◦C. The supernatant was separated from the mixture by centrifugation at 14,000 rpm for 5 min, and excess protein in the supernatant was estimated using the Bradford method by UV–Vis spectroscopy (Infinite F200 PRO, Tecan Group Ltd., Mа¨nnedorf, Switzerland) at λ = 595 nm. Bio-Rad protein assay dye was employed for the evaluation of protein adsorption performance with the equation

$$Q = Q\_0 \left(\frac{I\_0 - I}{I\_0}\right) \tag{1}$$

where *Q* is the adsorption capacity of protein on peptide–HAp, *Q*<sup>0</sup> is the initial amount of protein, *I*<sup>0</sup> is the initial absorbance intensity in the supernatant, and *I* is the absorbance intensity of the supernatant following adsorption.

### *3.5. Calculation of Carboxyl Group Density in Peptide–HAp*

First, EDC (42.9 mg) and NHS (5.1 mg) were each dissolved in 3 mL of 10 mM phosphate buffer (pH 7.0), and the solutions (500 μL) were mixed together. One mg of peptide–HAp was added to the mixture and stirred for 3 h at 20 ◦C. The solid materials were separated by centrifugation at 14,000 rpm for 5 min and then washed three times with 10 mM phosphate buffer (pH 7.0). The precipitant was resuspended in the same phosphate buffer (500 μL), and then 500 μL of 5-aminofluorescein solution (8 μg mL−1) was added to the suspension. After stirring in the dark overnight at 20 ◦C, the solid materials were collected by centrifugation at 14,000 rpm for 5 min and washed with the aforementioned buffer. Finally, the carboxyl group density in the precipitant was redispersed in a 1 mL buffer and measured using a spectrofluorophotometer (RF-5300PC, Shimadzu Co., Kyoto, Japan), of which the excitation and emission wavelengths were 494 and 521 nm, respectively.

#### **4. Conclusions**

In summary, we designed two types of self-assembling peptides with different secondary structures: (leucine–glutamic acid–leucine–leucine)5-PEG70 (LELL) and (valine–glutamic acid–valine–valine)5-PEG70 (VEVV), and these peptides were used as templates for HAp biomineralization. Moreover, we also investigated the effect of secondary structures within peptide-template–HAp on the particles and protein adsorption behavior. It could be shown that as-synthesized peptide LELL or VEVV showed almost entirely α-helix or β-sheet contents within secondary structures, respectively. The morphologies of all peptide–HAp were similar to bare HAp, whereas VEVV–HAp displayed a slightly plate-like structure. Additionally, all peptide–HAp have pore sizes of 30 nm, which may be expected for enzyme stability on enzyme immobilization, as indicated in our previous study. Furthermore, for the adsorption properties of proteins, it was revealed that each peptide–HAp specifically adsorbed basic protein (i.e., Cyt c and LSZ). With increasing amounts of peptide, the blocking effects for proteins, except for basic protein, were also increased. Overall, the reason that VEVV–HAp (3 mg) with β-sheet structures exhibited increased Cyt c adsorption amounts compared with LELL–HAp (3 mg) containing α-helix structures is explained as follows: the carboxyl group density at the surfaces of VEVV–HAp (3 mg) was more than 2-times higher compared with LELL–HAp (3 mg) while the carboxyl group density of peptide–HAp incorporated 1 mg of peptide amount was lower than that of peptide–HAp (3 mg). From these results, it can be stated that synthesized HAp on a self-assembling peptide template could be useful as a carrier for protein immobilization in biosensing and bioseparation applications and as enzyme-stabilizing agents.

**Author Contributions:** Conceptualization, K.K.; Data curation, S.K. and H.N.; Formal analysis, S.K. and H.N.; Writing—original draft preparation, S.K.; Writing—review and editing, S.L., F.N., and K.K.; Funding acquisition, S.L., F.N., and K.K.

**Funding:** The financial support of Grants-in-Aid for Scientific Research (C) no.15K06474 from the Japan Society for the Promotion of Science and A-STEP (JPMJTS1624) from the Japan Science and Technology Agency is gratefully acknowledged.

**Acknowledgments:** The author thanks Hiroyuki Iwata (Aichi Institute of Technology) for his help in the STEM-EDX observation. The authors would like to thank MARUZEN-YUSHODO Co., Ltd. (http://kw.maruzen. co.jp/kousei-honyaku/) for the English language editing.

**Conflicts of Interest:** The authors declare no conflict of interest.
