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Bioconjugate Chem. 2001, 12, 684−690
On the Preparation, Characterization, and Enzymatic Activity of Fungal Protease-Gold Colloid Bioconjugates Anand Gole,† Chandravanu Dash,‡ Chinmay Soman,† S. R. Sainkar,† Mala Rao,‡ and Murali Sastry*,† Materials Chemistry and Biochemical Sciences Division, National Chemical Laboratory, Pune 411 008, India. Received October 5, 2000; Revised Manuscript Received February 15, 2001
We present herein details pertaining to the preparation of bioconjugates of colloidal gold with aspartic protease from the fungus Aspergillus saitoi (F-prot) and their characterization and enzymatic activity. Simple mixing of the colloidal gold and protein solutions under protein-friendly conditions (pH ) 3) followed by centrifugation (to remove uncomplexed gold nanoparticles and protein molecules) results in the formation of the fungal protease-gold nanoparticle conjugates. The protein-gold nanoparticle bioconjugate was redispersed in buffer solution and indicated the formation of efficient bioconjugates with intact native protein structures. The bioconjugates in solution were characterized by UV-vis spectroscopy, fluorescence spectroscopy, and biocatalytic activity measurements while drop-dried bioconjugate films on Si (111) substrates were characterized by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), and X-ray diffraction (XRD) measurements. Microscopy images do show some aggregate formation, but the intactness of the native structure of the enzyme in the bioconjugate material was verified by fluorescence and biocatalytic activity measurements. The enzyme retains substantial biocatalytic activity in the bioconjugate material and was comparable to that of free enzyme in solution.
INTRODUCTION
Colloid chemistry includes the study of a wide range of entities spanning micron-sized to nanometer-sized particles, the properties of the particles being vastly different from that of the bulk material. There has been a great deal of research in different areas of colloid chemistry, viz., protein chemistry, inorganic nanoparticles such as quantum dots, clay materials, interfaces, and membranes, to name a few. Combination of different branches of colloid chemistry is an important goal with exciting spin-offs expected in industrial, medical, and advanced materials applications. Potential applications in the food, chemical, and agriculture industries, markers in electron or standard light microscopy and medicine such as drug delivery systems, efficient immunosensing assays for early detection of diseases, etc., has seen an explosion in the research and design of a new class of materials commonly known as bioconjugates. Formation of bioconjugates by immobilization protocols into different 2D and 3D matrixes is currently a great challenge as far as the stability, accessibility, shelf life, transportation, cost, and ease of manufacturing etc. are concerned. An important requirement for immobilization is that the biomolecule in the bioconjugate material should remain in its natural form and should be able to carry out necessary functions as efficiently as in solution. To date, biomolecules such as proteins have been immobilized on/ within various supports such as phospholipid bilayers,1 self-assembled monolayers,2 Langmuir-Blodgett films,3 polymer matrixes,4 the galleries of R-zirconium phosphates,5 in hydrophobic controlled pore-glasses,6 and in * To whom all correspondence should be addressed. Ph: +91 20 5893044. Fax: +91 20 5893044/5893952. e-mail: sastry@ ems.ncl.res.in. † Materials Chemistry. ‡ Biochemical Sciences Division.
films of thermally evaporated fatty lipids.7 All these systems deal with single protein immobilization protocols. In a different approach, Caruso and co-workers have demonstrated that the layer-by-layer (L-B-L) method can be used for the formation of multilayer protein-polymer conjugates.8a,b In this method, electrostatic attraction between alternately deposited charged species of proteins and polyelectrolytes is used to form multilayer planar films. Studies on using colloidal particles as a versatile and efficient three-dimensional (3-D) template for the immobilization of biomolecules has been recognized since the early 1980s.9 Immunomicrospheres, as one class of conjugate-colloids are called, can be defined as specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces.9 Appropriately coated colloidal particles can be used to react in a very specific way with antigens, target cells or viruses, depending on the type of antigen adsorbed on the microspheres. Immunomicrospheres have opened up new opportunities in the development of techniques for disease diagnosis and therapy. A number of groups have studied the adsorption of proteins on both organic and inorganic colloidal particles. As an extension of planar surfaces, Caruso et al. have successfully extended the L-B-L technique to form multilayer protein layers on polymer colloidal systems.8c,d The multilayers of proteins on colloidal particles increases the density of biomolecules and thus, their sensitivity in different applications. The stabilization of colloidal particles of polymers by proteins has been studied by Molina-Bolivar et al.10 Covalent attachment of immunoglobulins to the surface of polystyrene latex colloidal particles has also been demonstrated.11 Schmitt et al.12 have reported on a monomolecular layer of bovine serum albumin (BSA) on the surface of polystyrene (PS) latex particles. Elgersma et al. have demonstrated the
10.1021/bc0001241 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/16/2001
Fungal Protease−Gold Colloid Bioconjugates
adsorption of BSA on positively and negatively charged polystyrene lattices.13 The interaction of noble metal colloidal particles such as gold and silver with proteins/enzymes as well as the study of the enzymatic activity of the nano-bioconjugates has also received attention. Natan and co-workers have studied in great depth cytochrome c-gold colloid conjugates as regards stability, protein orientation, and application in surface enhanced Raman scattering (SERS) and found that metal-protein-metal sandwiches offer benefits as reagents for protein SERS.14 Macdonald and Smith have studied the orientation of cytochrome c adsorbed on citrate-reduced silver colloids.15 Single molecule spectroscopy (SMS) of the hemoglobin molecule adsorbed on 100 nm sized citrate-reduced silver colloidal particles was achieved by SERS.16 Silver particle aggregation is necessary for this effect, and the protein molecules actually bind the Ag particle clusters together.16 In the area of metal nanoparticle-enzyme conjugate materials, Crumbliss, Stonehuerner, and coworkers have studied the formation and enzymatic activity of gold nanoparticles complexed with horseradish peroxidase17a and xanthine oxidase17b as well as glucose oxidase and carbonic anhydrase molecules.17c A salient feature of their work is the demonstration that enzyme molecules are bound tightly to gold colloidal particles and retain significant biocatalytic activity in the conjugated form while the enzyme molecules dentature on adsorption to planar surfaces of gold.17c As part of our ongoing investigation into the formation and catalytic activity of enzyme-bioconjugate materials,7 we investigate herein the immobilization of fungal protease (F-prot) molecules on 3-D supports such as colloidal gold particles, leading to the formation of efficient bioconjugates. We observe significant catalytic activity of the F-prot molecules in the F-prot-Au colloid bioconjugate material which indicates that the enzyme molecules are immobilized without distortion to the enzyme native structure. The bioconjugates of 35 Å colloidal gold particles with the enzyme F-prot are formed under enzyme-friendly conditions (at pH ) 3) where the enzyme shows optimum catalytic activity. The bioconjugate was prepared by simple addition of the F-prot molecules to the colloidal gold solution held at pH ) 3. Thereafter, the F-prot gold colloid solution was centrifuged to remove uncoordinated F-prot molecules in solution to yield the purified F-prot-Au colloid bioconjugate material. The purified F-prot-Au colloid bioconjugate material was resuspended in appropriate buffer solution after rinsing several times. UV-vis spectroscopy was used to monitor the surface derivatization before and after centrifugation/ resuspension. The bioconjugate material were further characterized by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), X-ray diffraction (XRD), fluorescence spectroscopy, and biocatalytic activity measurements. The amount of F-prot on the colloidal gold surface was quantified by fluorescence spectroscopy and used for the determination of specific activity of the enzyme. The specific activities of the enzyme on the surface of colloidal gold and that of free enzyme in solution are comparable, indicating that the enzyme in the bioconjugate is in its active natural conformation. Presented below are details of the investigation. EXPERIMENTAL SECTION
Chemicals. F-prot and hemoglobin were obtained from Sigma Chemicals and used without further purifi-
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cation. Chloroauric acid and sodium borohydride were obtained from Aldrich Chemicals and used as received. All buffer salts were from standard commercial sources and of the highest quality available. Colloidal Gold Synthesis. Colloidal gold was synthesized by the borohydride reduction of 1.25 × 10-4 M concentrated aqueous solution of chloroauric acid (HauCl4). This process yields 35 ( 7 Å sized colloidal gold particles as shown previously by us.18 The colloidal solution was aged for a period of 12 h. The ruby red colored solution yielded an absorbance maximum at 518 nm.18 Formation of F-prot-Colloidal Gold Bioconjugates. A 10-6 M standard solution of the enzyme, F-prot (molecular weight ∼ 37000, pI ∼ 9.5) was prepared in glycine‚HCl (0.05 M, pH ) 3) buffer. Different amounts of this standard solution were added to colloidal gold to yield an F-prot concentration of 10-7, 10-8, and 10-9 M in the conjugate solution. The pH of the colloidal gold solution was adjusted to 3 prior to addition of F-prot. This was done since F-prot shows optimum biocatalytic activity at pH ) 3. The solution was kept for a period of 12 h at 4 °C and then centrifuged to remove uncoordinated F-prot and free gold particles in solution. The powder so obtained was rinsed several times with buffer solution and then resuspended in the pH ) 3 buffer and stored at 4 °C for further experimentation. UV-Vis Spectroscopy. The formation of the F-protgold colloid bioconjugate was studied by UV-vis spectroscopy using a Shimadzu dual-beam spectrophotometer (model UV-1601 PC) operated at a resolution of 1 nm. The absorbance band above 500 nm arising from excitation of the surface plasmon resonance in the gold particles18,19 and the resonance at 280 nm due to the π-π* transition in the tryptophan and tyrosine residues of the enzyme20 were monitored immediately after addition of F-prot to the gold solution as well as after centrifugation/resuspension. Three separate sets of F-prot-gold colloid bioconjugate solutions were prepared and tested for the above UV-vis signatures in order to check the reproducibility of the data. Scanning Electron Microscopy (SEM) and Energy Dispersive Analysis of X-rays (EDAX). SEM and EDAX measurements were performed on a Leica Stereoscan-440 scanning electron microscopy (SEM) equipped with a Phoenix EDAX attachment. A drop-dried F-protgold colloid bioconjugate film (10-7 M F-prot in the bioconjugate solution) on Si (111) substrate was used for the analysis. SEM analysis was primarily used to observe the morphology of the bioconjugates in the film. X-ray Diffraction Studies (XRD). A few drops of the centrifuged/resuspended F-prot-gold colloid bioconjugate solution were placed on 2 cm × 2 cm Si (111) substrates and dried in air. XRD measurements of these films were made on a Philips PW 1830 instrument operating at 40 kV voltage and a current of 30 mA with Cu KR radiation. The size of the colloidal gold particles in the bioconjugate film was estimated from the peak width of the (111) Bragg reflection using the Debye-Scherrer formula.21 Tertiary Structure Studies. Fluorescence spectroscopy is a powerful tool to study the tertiary structure of proteins and enzymes. The fluorescence of the F-protgold colloid bioconjugate solution was studied using a Perkin-Elmer Luminescence Spectrometer (model LS 50B). The tryptophan and tyrosine residues in the enzyme were used for excitation at 280 nm, and the broad emission band having a maximum at about 336 nm was recorded.22 Fluorescence spectra of bare colloidal gold particles in water and that of the buffer used were also recorded as control experiments. As expected, these
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controls did not show any emission in the region of interest (ca. 336 nm). Estimation of Amount of F-prot in the Bioconjugate Composite System. The fluorescence emission intensities at ca. 336 nm22 due to excitation of tryptophan and tyrosine residues in the enzyme (absorption at 280 nm) were recorded for different concentrations of the free enzyme in buffer solution, and a calibration curve was obtained. The amount of F-prot in the different bioconjugate solutions prepared in this study was estimated by comparing the intensity of fluorescence emission at 336 nm in the bioconjugate solution with the calibration curve for this protein. The amount of F-prot estimated in this fashion was then used to calculate the specific activity of the enzyme in the bioconjugate solution. Biocatalytic Activity Measurements. The biocatalytic activity of the centrifuged/resuspended F-prot-gold colloid bioconjugate solution was determined by reaction with a solution of hemoglobin (5 mg/mL) prepared in glycine‚HCl buffer at pH ) 3. F-prot is a proteolytic enzyme which acts on proteins such as hemoglobin23 and has been successfully used by us in studies on thin films of the enzyme immobilized in fatty lipid films.7b The enzyme fungal protease is not a well-studied system, and consequently information on specific chromogenic or fluorogenic substrates is not available. This has prompted us to use hemoglobin as a substrate in estimating the specific activity of the enzyme in the F-prot-gold colloid bioconjugate solution and of the free enzyme in solution. In a typical experiment to estimate the biocatalytic activity of the bioconjugate solution, 1 mL of the solution consisting of 100 µL of the F-prot-gold colloid bioconjugate material and 0.9 mL of the buffer was reacted with 1 mL of the hemoglobin solution, and the reaction mixture was incubated at 37° C for 30 min. After the incubation time, 1.7 M perchloric acid was added to the reaction solution to precipitate the remaining hemoglobin. After 30 min, the precipitate was removed by centrifugation and the optical absorbance of the filtrate measured at 280 nm. F-prot digests hemoglobin and yields acid soluble products (tryptophan and tyrosine residues) which are readily detected by their strong UV signatures at 280 nm.20 From the amount of F-prot estimated from the fluorescence studies, the specific activity of the enzyme was calculated. For comparison, the biocatalytic activity of similar concentration of free F-prot in solution was recorded. To check the reproducibility of the biocatalytic activity measurements, 10 separate measurements of the F-prot-gold colloid bioconjugate material were performed as mentioned above, and the average specific activity was calculated. RESULTS AND DISCUSSION
The choice of colloidal gold in the formation of bioconjugates is dictated by a number of advantages which these particles display. The striking ruby red color of colloidal gold makes it an ideal medium for colorimetric determination of molecular recognition processes as has been demonstrated for the protoypical biotin-avidin interaction.24 Colloidal gold has been used extensively by Mirkin, Letsinger and co-workers in the immobilization of oligonucleotides and detection of complementary base sequences by changes in the visible absorption spectra.25 The surface of colloidal gold does not require modification for chemisorption of proteins/enzymes as in the case of organic colloids. The ease of characterization of colloidal gold by observing colorimetric changes upon immobilization of analytes from solution, its biocompatibility, the
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Figure 1. (A) UV-vis spectra of the colloidal gold solution before (curve 1) and after addition of 10-6 M F-prot to the colloidal gold solution at pH ) 3 (curve 2) to give an equilibrium concentration of 10-7 M of F-prot in the bioconjugate solution. (B) UV-vis spectra of F-prot-gold colloid bioconjugate solutions after centrifugation, rinsing and resuspension in pH ) 3 buffer. The spectra corresponding to concentrations of F-prot in the starting bioconjugate solutions are curve 1-10-7 M, curve 2-10-8 M, and curve 3-10-9 M.
stability of enzymes on colloidal gold particle surfaces (as also observed by Stonehuerner et al),17 the simplicity of bioconjugate formation by just addition of proteins/ enzymes to the colloidal gold solution, and the fact that the surface does not require premodification has led to the choice of colloidal gold in the formation of bioconjugates of F-prot in this study. Preparation of F-prot-Colloidal Gold Conjugates. The as-prepared pH of colloidal gold by the borohydride reduction procedure is ca. 9.15 The enzyme, F-prot, has a pI of about 9.5 and shows optimum catalytic activity at pH ) 3,7b and hence the pH of colloidal gold has to be lowered prior to addition of F-prot. As mentioned in the Experimental Section, 10-6 M standard solution of F-prot was prepared in glycine‚HCl (0.05 M, pH ) 3) buffer and added to colloidal gold solution at pH ) 3, to form equilibrium F-prot solution concentrations of 10-7, 10-8, and 10-9 M, respectively. It is wellknown that thiol groups bind to the gold surface via a covalent bond.18 Cysteine residues on the surface of F-prot molecules may thus bind to the colloidal gold particles via a thiolate linkage. Support for cysteinemediated binding of enzyme to the gold colloids comes from the report of Sasaki et al.26 who demonstrated that the functional protein, myosin subfragment 1, binds to gold thin films through an Au-S bond involving cysteine residues in the protein. It has been reported in the literature that alkylamine molecules form a weak covalent bond with colloidal gold surfaces.27 Natan and coworkers indicate the role of the NH2 group in lysine rich pockets for the binding of proteins to the colloidal gold surface.14 We believe that the binding of fungal protease to colloidal gold is covalent in nature and occurs either via the cysteine or lysine residues or a combination of both. The number of cysteine and lysine residues in the enzyme capable of binding to the gold particle surface would require detailed information regarding the amino acid sequence in F-prot which is not available at the moment. Figure 1A shows the UV-vis spectra recorded from the gold colloidal solution before (curve 1) and after (curve 2) addition of the F-prot solution (overall F-prot concentration in the solution ) 10-7 M). The spectra have been displaced vertically for clarity. The surface plasmon resonance due to gold can be clearly seen at about 526
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Figure 2. (A) SEM image of a film of the F-prot-gold colloid bioconjugate material drop-dried on a Si (111) substrate (see text for details). (B) SEM image of an aggregate of F-prot and gold colloidal particles in a drop-dried film of bioconjugate material at higher magification (see text for details).
nm18 for the as-prepared gold solution (curve 1, Figure 1A). On addition of F-prot, a broadening and red shift of the plasmon resonance is seen (curve 2, Figure 1A) and clearly indicates surface coordination of F-prot with the gold particles. The broadening of the plasmon resonance is also indicative of some degree of aggregation of the gold particles mediated by the protein molecules. The absorbance at about 280 nm seen prominently in the F-protgold colloid bioconjugate solution (curve 2, Figure 1A) is due to the π-π* transitions in the tryptophan and tyrosine residues in the enzyme.20 The spectra for other concentrations of F-prot (10-8 and 10-9) gave similar features and are not shown for brevity. The bioconjugate solutions were stored for a period of 12 h at 4 °C before centrifugation and resuspension. UV-vis spectra of the F-prot-gold colloid bioconjugates recorded after centrifugation, rinsing, and resuspension in water for 10-7, 10-8, and 10-9 M F-prot in the starting bioconjugate solution are shown as curves 1-3, respectively, in Figure 1B. It is observed that both the surface plasmon resonance intensity from the gold particles as well as the concentration of F-prot molecules in the resuspended bioconjugate solution (as evidenced by the intensity of the absorption at 280 nm) scale with the concentration of F-prot molecules in the starting solution (i.e., the solution prepared prior to centrifugation). Thus, a larger number of gold particles are required to stabilize a greater concentration of F-prot molecules in solution and provides a simple recipe for increasing the loading factor of the enzyme molecules in the bioconjugate material. The magnitude of the absorbance at 280 nm in the case of 10-7 M F-prot-gold colloid bioconjugate material before centrifugation is about 0.3 absorbance units which is reduced to 0.06 absorbance units after centrifugation and resuspension. This is also true for other concentrations of F-prot in the bioconjugate material and indicates that the large decrease in intensity of this band is due to removal of uncoordinated F-prot molecules from the respective solutions. Three separate experiments each of bioconjugate formation, rinsing, and resuspension for the three concentrations of F-prot in the bioconjugate material were performed to check the reproducibility of the data. It was found that the curves and the absorbance at 280 nm (corresponding to that of F-prot in the
bioconjugate solution) agreed in the experiments to within 10%. SEM and EDAX Measurements. Figures 2A and B show the SEM images of F-prot-gold colloid bioconjugate films formed by drop-drying the bioconjugate solution (10-7 M F-prot in the bioconjugate solution) on Si (111) substrates. A number of aggregates can be seen in the lower magnification image (Figure 2A) and is likely to be due to enzyme-induced cross-linking of the colloidal gold particles. Figure 2B gives a magnified view of one of the aggregates. On careful observation, it is seen that the aggregate is made up of dark and bright spots. EDAX spot measurements were performed, and the dark regions were found to be deficient in gold particles (but enhanced in protein concentration as evidenced by a large nitrogen signal) while the bright regions were rich in gold particles. Thus the SEM images indicate aggregates within the F-prot-gold colloid bioconjugate material with spatially separated regions of gold particles and protein molecules. We would like to add here that a similar surface morphology was observed by Caruso et al. in multilayer films of a polymer-anti-IgG composite material.28 As mentioned above, EDAX measurements were performed on the above film and the average atomic percentage ratio of gold: nitrogen over the surface of the film was estimated to be 1:5 and that of gold:carbon was recorded to be 1:14. The nitrogen and carbon signals arise from the enzyme molecules and serve as a good indicators for the presence of enzyme in the F-prot-gold colloid bioconjugate material. XRD Measurements. While formation of aggregates in the F-prot-gold colloid films is clearly seen in the SEM pictures (Figure 2), it was difficult to image the individual gold particles in the aggregates due to limited resolution of the instrument. It is well-known that the size of particles in such nano-structured films may be estimated from the line-broadening of Bragg reflections in the XRD patterns using the standard Debye-Scherrer formula.21,29 A few drops of the centrifuged/resuspended F-protcolloidal gold bioconjugate solution (prepared from the 10-6 M F-prot starting solution) were placed on 2 cm × 2 cm Si (111) substrate and dried in air. Figure 3A shows the XRD pattern recorded from an F-prot-gold colloid
688 Bioconjugate Chem., Vol. 12, No. 5, 2001
Figure 3. (A) XRD pattern of a film of the F-prot-gold colloid solution on a Si (111) wafer prepared by a simple drop-drying method (see text for details). The solid line is a Lorentzian fit to the spectra. (B) Fluorescence spectra of F-prot solution at a concentration of 10-6 M at pH ) 3 (curve 1), F-prot-colloidal gold bioconjugate solution at pH ) 3 prepared from the 10-6 M concentration starting F-prot solution (curve 2) and F-prot solution at a concentration of 10-7 M at pH ) 3 (curve 3). Fluorescence spectra from glycine‚HCl buffer (0.05 M, pH ) 3, curve 4), and bare gold colloidal solution (curve 5) as controls are also shown (see text for details).
bioconjugate film deposited on Si(111) wafer by dropdrying the 10-7 M F-prot bioconjugate solution. The (111) Bragg reflection from gold is clearly seen at a 2 θ value of ca. 38.2° and has been fit to a Lorentzian (solid line, Figure 3A) to estimate the peak broadening. The size of the gold particles in the F-prot-gold colloid bioconjugate film was calculated from the fit to be 250 Å. The size of as-prepared colloidal gold particles is about 35 Å,18 but on forming a bioconjugate with F-prot molecules, the size increases indicating a small amount of sintering of the particles. However, given that the enzyme on the surface of the colloidal gold particles is active (as explained subsequently in the section on biocatalytic activity measurements), enzyme aggregation may be ruled out. Tertiary Structure Studies. The biocatalytic activity of the immobilized enzyme would, to a large extent, depend on the tertiary structure of the enzyme remaining unperturbed on the surface of colloidal gold. The tertiary structure of an enzyme can be checked monitoring the fluorescence emission from the tryptophan or tyrosine residues in the enzyme. This is a standard procedure, and we have used it to check the tertiary structure of F-prot on the surface of colloidal gold by excitation of the π-π* transition in the tryptophan and tyrosine residues at 280 nm.22 Figure 3B shows the fluorescence spectra recorded from 10-6 M and 10-7 M aqueous solutions of the free enzyme, F-prot, at pH ) 3 (curves 1 and 3) as well as the fluorescence spectrum measured from the F-prot-colloidal gold bioconjugate solution prepared from the 10-6 M F-prot starting solution (curve 2). The intensity of absorbance maxima in all three solutions occurs at about 336 nm which indicates that the F-prot molecules in the bioconjugate solution are present in their natural configuration maintaining their tertiary structure. Therefore, even though aggregates were seen in the SEM images, fluorescence measurements show that the enzyme molecules in the aggregates maintain their tertiary structure. Control experiments were performed on the fluorescence from the glycine‚HCl (0.05 M, pH ) 3) buffer (Figure 3B, curve 4) and the bare colloidal gold solution (Figure 3B, curve 5) by excitation at 280 nm. It is observed that for these two controls, there is no
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emission in the spectral region where the enzyme contributes (ca. 335 nm) to the signal. This result is important in obtaining a reliable estimate of the concentration of F-prot molecules in the redispersed F-protgold colloid bioconjugate solution, as discussed below. The fluorescence spectroscopy measurements of Figure 3B were used to quantify the amount of F-prot on the colloidal gold surface in the bioconjugate solution by comparing the intensity of the emission signal at 336 nm with the standard calibration curve. The calibration curve was obtained by plotting the fluorescence intensities of different concentrations of F-prot at pH ) 3. The concentration of F-prot in the F-prot-gold colloidal bioconjugate system was calculated to be ca. 3.7 × 10-7 M and is an important number in the estimation of the specific activity of the bioconjugate solution and enables comparison of the activity of the enzyme in the bioconjugate material with that of the free enzyme in solution. Biocatalytic Activity Measurements. The most important aspect in protein-colloid bioconjugate systems is the retention of the biocatalytic activity after adsorption onto the nanoparticle surface. A 100 µL volume of F-prot-gold colloid bioconjugate solution (starting F-prot concentration in the bioconjugate material ) 10-6 M) was reacted with hemoglobin as explained in the Experimental Section. The specific activity was calculated using the amount of enzyme estimated from the calibration curve (see Figure 3B and discussion above) and compared with that of free enzyme in solution under identical conditions. The specific activity of free enzyme in solution was 6.1 units, and that of the enzyme immobilized onto colloidal gold surface was 5.5 ( 0.4 units. (One unit of enzyme will produce a change in absorbance at 280 nm of 0.001 per minute at pH ) 3.0 and 37 °C measured as acid soluble products using hemoglobin as the substrate).7 The value quoted for the specific activity of the enzyme in the bioconjugate solution is a statistical average over 10 separate measurements of freshly prepared solutions of the same concentration F-prot molecules in the bioconjugate. The small standard deviation shows that the results are highly reproducible. A comparison of the specific activities of the free enzyme and enzyme in the bioconjugate solution shows that there is not much decrease in the activity of the enzyme molecules in the bioconjugate system. This is a significant result and shows that the enzymes in the aggregates have not lost their biocatalytic activity and are possibly stabilized by the gold nanoparticles. Conclusions. F-prot-gold colloid bioconjugates have been prepared by simple addition of F-prot molecules to the colloidal gold solution. A microscopic analysis of the bioconjugate material showed the presence of aggregates of the enzyme, possibly cross-linked and stabilized by the gold particles. Even though aggregation of the protein was indicated, fluorescence and biocatalytic activity measurements clearly showed that the enzyme on the surface of colloidal gold retains its native confirmation and furthermore, that the specific activity of the enzyme in the bioconjugate was comparable to that of the free enzyme in solution. ACKNOWLEDGMENT
Two of us (A.G. and C.D.) thank the Council of Scientific and Industrial Research (CSIR), Government of India, for financial assistance.
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