UV–FT-IR Studies on α-Lactalbumin-Infused Nickel Hydroxide Nanoparticles

The merits and defects in using the mineral nickel in human beings are mentioned. Efforts are continuously made in the research to ensure the safe use of this mineral by various field experts. The present work is also a part of one such trial. The chemical co-precipitation method is used for the synthesis of the nickel hydroxide (Ni(OH)₂) nanoparticles. Nano-sized α-lactalbumin particles are blended with the Ni(OH)₂ nanoparticles using heat processing. Analysis of this final product by UV and FT-IR reveals that the so-called final product is found to be α-lactalbumin-infused Ni(OH)₂ nanoparticles. It is peculiar to note that though the α-lactalbumin is a macromolecule, it became infused into the Ni(OH)₂ layers, and the existing structural and functional variations in the systems are reflected in the observed parameters. The cumulative effects of various electrostatic interactions existing in the system are related to the observed structural patterns. The further scope of the said nanoparticle in the application area in various fields was identified.

One of the many transition elements that are extensively found in the air, water and soil or in general in our environment, is nickel (Ni). It is ubiquitous, silver-like in appearance and freely available in the earth.¹ Among the various oxidation states of nickel, the +2 state of [Ni(II)] is comparatively more stable in biological systems.^2,3

In this day-to-day busy modern world, the use of nickel and its many alloys is inevitable.⁴ In case of human beings, the use of nickel has beneficial as well as harmful effects. Almost every human usage of nickel is in trace level and any form of excess usage is ultimately hazardous.

The healthy development of animals (including human) and plants, even the aqueous and geographic microbes all essentially need nickel as one of the essential elements.^5–7 It is often noticed that a deficit in the level of nickel in bio systems seriously disrupts the intra-uterine growth and leads to anemia.⁸ Min, Cha, and Kang⁹ have commented that Ni is highly essential for the proper functioning of many human enzymes like urease and hydrogenase. Right from enzymes up to DNA, the positive and negative roles of nickel were detailed by many authors.^10–12

Apart from direct consumption, even the exposure to nickel-rich environments are found to have serious pathological effects in humans.^2,13–15 In view of all these unwanted hazardous effects caused by nickel to human systems, it was declared as the “Allergen of the Year” in 2008.¹⁶

All these facts have resulted in a strong dilemma regarding the medicinal usage of nickel, that too in in vivo condition. However, the recent developments in nanotechnology and drug delivery seem to provide a solution for this problem that is these advancements have discovered the very safe-use methodology of nickel in therapeutics. As per this procedure, the toxic mineral nickel is blended/infused/embedded/mixed with any or some biocompatible material and its toxicity has been drastically restricted. Glennon and Sarkar¹⁷ have reported that in human blood circulation system, nickel has been transported mainly by globular proteins such as albumins.¹⁸ The interactions of such proteins with nickel are so remarkable that the ionic interactions of nickel are drastically reduced. Hence, it is possible for any of the biocompatible macromolecule to bind this mineral particle so as to lessen its toxic effects and can carry the particle to the desired site for which the present study aims to analyze the capability of globular protein α-lactalbumin. In this work, the term lactalbumin (or LA) always refers to α-lactalbumin.

While progressing the proposed task few prime questions arise in mind that i) whether the final product is lactalbumin infused Ni(OH)₂ nanoparticle or Ni(OH)₂ infused lactalbumin nanoparticle; ii) whether the cumulative interactions of the final product be beneficial or harmful to human system; and iii) is it viable to use them in humans under in vivo conditions?

Of the two components, the lactalbumin and the nickel, nickel is a tinier molecule and lactalbumin is a macromolecule. Hence, superficially Ni(OH)₂ infused lactalbumin as the final product is highly logical and the lactalbumin infused Ni(OH)₂ is not logically possible. Further, in the former case, Ni(OH)₂ is totally screened off so that its beneficial effects may be unavailable. But, in later cases, Ni(OH)₂ forms the outer layer and its toxicity may be more. So, it is necessary to ascertain safe use under in vivo conditions and how its toxic effect can be minimized. All such uncertainties are attempted in this work.

Thus, the intention of the present work is to synthesize the said nanoparticle and analyze for its infusion structure and the cumulative interactions which is expected to provide a clear guidance in using this particle for human therapeutic usage.

Materials and Methods

Synthesis of Experimental Nanoparticles

Nickel hydroxide nanoparticles were prepared by co-precipitation method as depicted in Figure 1. All the chemicals (AR grade) used were purchased from Aldrich. A precursor solution of 0.1 M nickel nitrate (NiNO₃.6H₂O) was prepared initially. Another solution containing (0.25, or 0.5, or 0.75, or 1 mM of) α-lactalbumin mixed in 0.1 M of sodium hydroxide was added to the precursor solution in a drop-wise manner with continuous stirring.

The temperature of the setup was kept at 70 °C and the pH was maintained at 8.4 by using monobasic and dibasic sodium dihydrogen phosphate buffer. A Hanna pH meter (microprocessor-controlled), supplied by Hanna Instruments, was used to measure the pH values of all solutions. After 4 hours, the solution was washed with distilled water and centrifuged repeatedly. The Eppendorf vials were centrifuged for 10 minutes at 15,000 rpm and 20 °C using a Hettich centrifuge (Germany). The final precipitate was dried, powdered, and subjected to dynamic light scattering (DLS) method that confirmed the formation of the nano-sized final particles and was used for the analytical characterization of the present work. The same particles were dissolved in 5% ammonia solution for recording data in UV-Visible study.

UV-Visible Study

UV-visible spectra were recorded using a Jasco UV–Visible double-beam spectrophotometer (Model No. V-750) with 1 cm quartz cuvette in the Centralized Instrumentation and Services Laboratory (CISL) of Annamalai University, TN, India. Double-distilled water, used for the preparation of solutions, was taken as the solvent for the UV spectrum of all the samples.

FT-IR Study

FT-IR primary spectra of all the experimental powdered samples were recorded using a Fourier transform infrared (FT-IR) spectrophotometer (Shimadzu IRSpirit-X) in the Department of Physics, Annamalai University, TN, India. The KBr pellet method was employed in transmittance mode, with a resolution of 1.0 cm^-1 over the 4000 cm^-1 to 450 cm^-1 range.

Determination of secondary structure reflects the behavioral nature of the protein involved. Protein FT-IR spectrum has many specific regions of which amide I region is very sensitive for secondary structural studies.¹⁹ Double derivation can be done using the Lorentzian function, which is a built-in option of the Origin 7.0 program. The program itself can identify, optimize and determine the relative area of the secondary structure peaks. For clarity, a few peaks were highlighted. Structural counts were obtained by consolidating the peaks, and assignments were made by comparison with the literature.^20,21

Results and Discussion

UV-Visible Study

The recorded UV-visible spectra of the chosen samples are shown in Figure 2. The spectra have their own characteristic peaks, which were compared with the available literature and are summarized in Table 1. Inspection of Table 1 reveals good agreement between the experimental and literature data, thereby affirming the availability of the individual components and the extent of their purity.

The key factors that decide the structural arrangements of the species in UV-Visible spectrum include the molecular conformation, molecular conjugation, the extent of saturation and also the morphology, size, atomic or molecular weight. However, factors like solvent effects and pH changes, among others, are always said to be the sources of the shift in absorbing wavelength.²⁷

Table 1 also lends validity to the experiments carried out. The ammonia (NH₃) peak in Figures 2(a) to 2(d) is around 210 nm and any slight shift is attributed to the molecular conformation of ammonia.²² Figures 2(b) and 2(d) show that, for α-lactalbumin, the UV has an absorbance nearing 286 nm. This absorption is wholly attributed to the combined strengths of conjugative transitions like the pi to pi* transitions (π–π^*) in tyrosine, tryptophan and phenylalanine of α-lactalbumin.^25,28,29 However, with a gradual increase of protein (or reduction in metal hydroxide), the protein peak intensity at 286 nm increases with a blue shift. This reveals that the interactions of nickel hydroxide with protein increasingly intensify, and the aromatic residues of protein are exposed toward polar environments. The change in protein peak intensity seems to depend on metal hydroxide concentrations. Further, the observed UV-vis results suggest that the increase in absorbance in response to the increase in protein is due to the complex formation between lactalbumin and the nickel hydroxide.

Figures 2(c) and 2(d) show the UV-vis absorption peaks for nickel hydroxide (Ni(OH)₂) particles and they are found around 368 nm (UV) and 592 nm (visible). Both of these peaks are attributed to d-d electronic transitions within the nickel ion (Ni²⁺) in its octahedral coordination with oxygen atoms.^30–32

Mutual comparisons of the formed peaks in the spectra offer valuable information in knowing and analyzing the actual picture that exists in the system and to account for the extent of infusion of α-lactalbumin. The perusal of Figure 2(a) with that of Figure 2(b) reveals a red shift of 7 nm to ammonia due to α-lactalbumin, whereas the perusal of Figure 2(a) and 2(c) indicates that Ni(OH)₂ also offers a red shift but by 5 nm to ammonia.But the inspection with Figure 2(d) is highly important because it reveals the predominance of metal hydroxide over lactalbumin by offering a uniform red shift of the same 5 nm to ammonia irrespective of α-lactalbumin content.Additionally, it is worth noting that Ni(OH)₂ exerts the same uniform red shift of a few (1 to 4) nm to lactalbumin.This uniform effect to two different species by a 3^rd component (Ni(OH)₂) is possible only if this third component lies between the first two species.Thus, the presence of Ni(OH)₂ as an outer layer to α-lactalbumin in the solvent ammonia is suggested by the observed UV features.It is also to be noted that the increase in the content of α-lactalbumin offers a continuous blue shift in UV-vis peaks.This may be attributed to the readily available free Ni²⁺ ions in the medium that can show d-d transitions.A similar scheme of protein intercalation between the brucite layers due to anionic exchange interaction was suggested by Yu et al.³³ and lends support to the present view of protein intercalation.

FT-IR Study

Figure 3 depicts the primary FT-IR spectra of all the experimental samples. The characteristic peaks in each FT-IR spectrum were identified, verified, and listed in Table 2. It summarizes the wavenumber, assignments and the type of vibration for both protein and metal hydroxide.

Specific peaks of α-lactalbumin at 1652 cm^-1 and 1532 cm^-1 and Ni(OH)₂ at 622 cm^-1 confirm the purity of the samples taken.^41–43 Peaks of the individual components were matched by identifying them in the spectra of the combination systems (Table 3).

Interpretation of the IR spectrum is highly region-sensitive with well-defined wavenumbers. In the present case one is lactalbumin having specific amide I pattern and the other is nickel hydroxide supposed to have its fingerprint patterns. However, in the combination systems, a slight shift in wavenumber is noticed. The shifts in the prominent peaks are summarized in Table 3. Interesting facts can be obtained from the perusal of this table. It is to be noted that the shift in one component due to the other is more or less the same and uniform and is toward the red-shift side for both cases. It indicates that the structural patterns are slightly but similarly affected in both.

Changes in structural patterns, such as compactness, texture, bonding mechanism, to almost the same extent for a macromolecule and a micromolecule are somewhat peculiar. This is possible only if either the macromolecule exists in a split-up manner or micromolecules appear in aggregate form and in the present case the latter possibility is given more chances due to their predominant ionic interactions. Hence, the Ni(OH)₂ molecules are supposed to form a sheet-like continuous cluster and the α-lactalbumin structure is much folded by Ni(OH)₂ and becomes denatured.This suggests the formation of intercalation of protein into the metal hydroxide sheets. Thus, the system is aptly termed the α-lactalbumin-infused Ni(OH)₂ system as initially pointed out in UV studies.

Any observed increase in the wavenumber of a peak is attributed to the corresponding increase in transition energy. Hence, the stretching at 1652 cm^-1 (amide I) or bending at 1532 cm^-1 (amide II) is possible at a still higher energy, and this indicates that the internal arrangements are weakened due to nickel hydroxide. A disintegrated form of α-lactalbumin and in less compact nature is quite logical and this is again analyzed by another method such as secondary structure analysis in the subsequent section.

It is important to consider the interaction of metal hydroxide. The red shift exhibited by the prominent peak at 622 cm^-1 indicates deformation in Ni(OH)₂. The increase in wavenumber implies an increase in the transition energy and hence leads to major modifications in structure. The matrix formation in Ni(OH)₂ spreads over the disintegrated protein and hence the system may resemble that shown in Figure 4. A similar opinion was suggested by Ngew et al.⁴⁴ in studies of layered double hydroxides.

As stated above, secondary structural analysis was attempted for the disintegrated, less compact form of the protein α-lactalbumin. The amide I region in the range 1610 cm^-1 to 1690 cm^-120,45 is the most responsive region for protein secondary structure analysis. The unique functional group in the amide I region is C=O³⁶ and it is found in the present primary spectra. The fingerprint frequencies that were found below 1500 cm^-1 indicates the molecule as a whole and not the individual atoms or bonds.

The amide I region is highly specific for sensing the changes in the arrangement of molecules or in bonding alignments as noted by Walter et al.⁴⁶ Hence, the present work utilizes that region. Specific peaks for protein appear at 1654 cm^-1 and 1656 cm^-1 along with non-prominent broad bands. These non-prominent broad bands are instrumentally irresolvable. Hence, to solve them effectively an appropriate mathematical technique must be adopted. The second-derivative curve-fitting method⁴⁷ is one method adopted here to comprehend and appreciate the secondary structure band components.

Figures 5(a) to 5(e), generated using the Origin program for the chosen combinations, depict the second derivative and the peak-sum trend. A supplementary file is also provided that contains the complete second-derivative images and the optimized second-derivative peaks of all the chosen α-lactalbumin systems. Details of commonly available secondary patterns are listed in one of our previous papers.¹⁹ For α-lactalbumin protein, most commonly available secondary patterns are α-helix and β-sheets. The α-helix exists at 1630 cm^-1 and in the range 1648 to 1660 cm^-1, whereas β-sheets exist in the range of 1612 to 1641 cm^-1 and in 1670 to 1694 cm^-1 range. Moreover, the same list shows that the 1640 to 1650 cm^-1 region is for random coils, 1660 to 1666 cm^-1 is for 3₁₀ helices and 1662 cm^-1 to 1684 cm^-1 is for β-turns. However, these are not so common for α-lactalbumin.

Tables 4 and 5 offer valid points for the structural stability and its type. This can be obtained from the consideration of all the interactions that are existing in the protein and the changes therein due to the metal hydroxide. It can be inferred from Table 5 that in the 0.25 mM α-lactalbumin, the contribution of α-helix is 33% whereas that of β-sheets is 60%. The presence of a greater number of β-sheets compared to the α-helix content indicates decreased stability due to the interaction of nickel hydroxide.

The native state of the protein is always supposed to be more in α-helix and less in β-sheets as noted by Permyakov et al.⁴⁸ Inspection of Table 5 clearly indicates that an increase in β-sheets with a decrease in α-helix content is observed due to the presence of Ni(OH)₂. Thus, Ni(OH)₂ weakens the structural stability of α-lactalbumin.

Likewise, assignments can be made for the other combinations of α-lactalbumin, and Table 5 lists the consolidated qualitative measurements. This table provides much valuable information.

α-lactalbumin is a peculiar protein in the sense that it is amphipathic.⁴⁹ Hence, the hydrophobic part of the protein is highly compressible, which means that numerous voids and vacant sites are available, as evidenced by the increasing β-sheets in various mM combinations. Above the isoelectric point, α-lactalbumin becomes anionic⁵⁰ and is therefore able to pass through charged layers. Hence, the chances for intercalation are quite high for this protein. The combined bonding network due to the weak van der Waals, hydrogen-bonding, and guest-host interactions⁴⁶ lend support to the uniform spreading and stability of the disintegrated protein into the molecular matrix of metal hydroxide. In other words, the aqueous phase of the protein is exposed by the electrostatic interactions between the positive groups (NH³⁺) and the negative groups (–COO^-) with the Ni(OH)₂ layers.⁵¹

Of the various secondary structures of proteins, random coils are also somewhat common.⁵² However, as cited earlier, due to the predominance of α-helix and β-sheets in lactalbumin, random coils are scarcely available, and hence their interactions with metal hydroxide are poor.

It is mentioned that β-sheets are the major source of intermolecular interactions, whereas the intramolecular interactions are due to α-helices.¹⁹ In the present work, the α-helix content is very low, and hence intramolecular interactions are highly remote, thereby strengthening the intermolecular interactions. Thus, the internal structural disturbances are minimal, whereas interactions between the protein and metal hydroxide increase substantially, with the net result of lending support to the stability of the protein.

The two adjacent segments of antiparallel β-sheets are found to be connected by β-turns.⁵³ However, in the present case, no β-turns are found in the 0.25 mM of α-lactalbumin, whereas in other combinations they exist. Of course, β-turns are highly transitory and hence their weak interactions are considered to be a part of the integrated interactions. Further, the absence of β-turns indicates the absence of antiparallel β-sheet segments.

Smith et al.⁵⁴ have commented that like α-helix, 3₁₀ helices and random coils are supports for the stability of native protein, but they are highly flexible and weak. Many authors^54,55 have combined the counts and observations of random coils, 3₁₀ helices, and β-turns to offer an effective interpretation. It can be noticed that they support the protein's stability as a whole and any reduction in one pattern is compensated by the other patterns.

Conclusions

Major outcomes in the present work are:

• UV studies reveal that the α-lactalbumin exists between the nickel hydroxide and the ammonia molecules.
• FT-IR studies suggest that the protein α-lactalbumin became intercalated into the sheets of nickel hydroxide.
• Both studies support the infusion of α-lactalbumin into the sheets of nickel hydroxide.
• IR studies further reveal the chemical disruption of the protein structure by the nickel hydroxide.
• The interactions due to Ni(OH)₂ are found to be so strong that the quantity of protein mixed becomes immaterial.
• The combined bonding network by the weak van der Waals forces, hydrogen bonding, and guest-host interactions is the key factor in deciding the intercalation mechanism and the structural stability of the protein.

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