Evaluation of Fibroblasts Adhesion and Proliferation on Alginate-Gelatin Crosslinked Hydrogel

Due to the relatively poor cell-material interaction of alginate hydrogel, alginate-gelatin crosslinked (ADA-GEL) hydrogel was synthesized through covalent crosslinking of alginate di-aldehyde (ADA) with gelatin that supported cell attachment, spreading and proliferation. This study highlights the evaluation of the physico-chemical properties of synthesized ADA-GEL hydrogels of different compositions compared to alginate in the form of films. Moreover, in vitro cell-material interaction on ADA-GEL hydrogels of different compositions compared to alginate was investigated by using normal human dermal fibroblasts. Viability, attachment, spreading and proliferation of fibroblasts were significantly increased on ADA-GEL hydrogels compared to alginate. Moreover, in vitro cytocompatibility of ADA-GEL hydrogels was found to be increased with increasing gelatin content. These findings indicate that ADA-GEL hydrogel is a promising material for the biomedical applications in tissue-engineering and regeneration.     

The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.

A procedure using 2,4,6-trinitrobenzenesulfonic acid (TNBS) for the determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials is presented. The major modification from previous procedures is an extended TNBS reaction time to allow a stoichiometric reaction with amino groups. In addition, autoclave hydrolysis is used to assure sample dissolution for spectrophotometric measurements. The assay accuracy was evaluated by determining epsilon-amino groups of insulin and bovine albumin. The determinations differed from literature values by < or = 3.3%. The epsilon-amino group content of Type B gelatin was found to be 33.0 mol/gelatin molecule of 1000 residues and is in agreement with similar source gelatins and collagen. The coefficient of variation for determinations on all three materials was < or = 5.3%. The assay should be applicable to a broad range of proteinaceous materials.     

Characterization of Fish Gelatin at Nanoscale Using Atomic Force Microscopy

Atomic force microscopy (AFM) was used as a meaningful tool to characterize the nanostructure of gelatin from catfish (Ictalurus punctatus) skin. The gelatins extracted with pretreatments including acid pretreatment, alkaline pretreatment, and alkaline followed by acid pretreatment (optimized extraction conditions). The resulting gelatins were imaged using AFM and their nanostructure was studied. The AFM images showed that gelatin extracted with acid pretreatment had a coacervate structure while with alkaline pretreatment there were separate aggregates. Spherical aggregates and annular pores were observed in AFM images of gelatin with the optimized extraction conditions. AFM imaging of gelatin with a relative high concentration (0.5%) was successfully done and the results help researchers to understand gelatin structures at the nanoscale.     

蚕丝蛋白和明胶复合组织工程材料与肝细胞相容性的实验研究

目的：研究蚕丝蛋白-明胶三维材料支架对人永生化肝细胞系QZG贴附及增殖的影响。方法：采用四氮唑盐比色法（MTT）、细胞计数法检测QZG细胞在纯蚕丝生物材料上与在蚕丝蛋白-明胶复合材料上的增殖情况,用扫描电镜观察QZG细胞在两种三维生物材料上的贴附与增殖情况。结果：QZG细胞可以在蚕丝蛋白生物材料贴附及增殖,在引入明胶的蚕丝蛋白材料上细胞贴附更紧密,增殖更明显。结论：蚕丝蛋白与明胶复合材料支架具有良好的细胞贴附性能,通过改进在肝组织工程应用方面将具有一定应用前景。     

Pachytene spermatocyte proteins influence Sertoli cell function

Isolated Sertoli cells were cultured on MatrigelTM-coated Millipore filters in bicameral chambers. The Sertoli cells form confluent epithelial sheets that, by virtue of the Sertoli cell tight junctions, form transepithelial permeability barriers between the apical and basal domains of the cells. These Sertoli cells secrete metabolically labeled proteins in a polarized manner. Three peptides, P1 (pI = 4.5-5.0, MW = 70,000), P2 (pI = 4.5-5.0, MW = 50,000), and P3 (pI = 4.0-4.7, MW = 34,000) are secreted apically from the epithelial sheets of Sertoli cells and are not found in basal secretions from the same Sertoli cells. Pachytene spermatocyte-conditioned medium contains proteins released from the germ cells that are uniquely different from the Sertoli cell-secreted proteins. Addition of the pachytene spermatocyte-conditioned medium to the apical reservoir of the bicameral chambers over an epithelial sheet of Sertoli cells stimulated the synthesis and secretion of total protein, transferrin, and specifically induced peptides S1 and S2 from Sertoli cells. As controls, conditioned medium from 3T3 fibroblasts and round spermatids did not stimulate the Sertoli cells. Hence, the ability of pachytene spermatocyte proteins to induce specific Sertoli cell secretion indicates that the pachytene spermatocytes are able to influence their surrounding milieu, and provides further support to the concept of a paracrine interaction between germ cells and Sertoli cells during spermatogenesis.     

Extended Latanoprost Release from Commercial Contact Lenses: In Vitro Studies Using Corneal Models

In this study, we compared, for the first time, the release of a 432 kDa prostaglandin analogue drug, Latanoprost, from commercially available contact lenses using in vitro models with corneal epithelial cells. Conventional polyHEMA-based and silicone hydrogel soft contact lenses were soaked in drug solution ( solution in phosphate buffered saline). The drug release from the contact lens material and its diffusion through three in vitro models was studied. The three in vitro models consisted of a polyethylene terephthalate (PET) membrane without corneal epithelial cells, a PET membrane with a monolayer of human corneal epithelial cells (HCEC), and a PET membrane with stratified HCEC. In the cell-based in vitro corneal epithelium models, a zero order release was obtained with the silicone hydrogel materials (linear for the duration of the experiment) whereby, after 48 hours, between 4 to 6 of latanoprost (an amount well within the range of the prescribed daily dose for glaucoma patients) was released. In the absence of cells, a significantly lower amount of drug, between 0.3 to 0.5 , was released, (). The difference observed in release from the hydrogel lens materials in the presence and absence of cells emphasizes the importance of using an in vitro corneal model that is more representative of the physiological conditions in the eye to more adequately characterize ophthalmic drug delivery materials. Our results demonstrate how in vitro models with corneal epithelial cells may allow better prediction of in vivo release. It also highlights the potential of drug-soaked silicone hydrogel contact lens materials for drug delivery purposes.     

Physico-chemical and antigenic characterization of unconventional heavy chain antibodies of Indian desert camel (Camelus dromedarius L.)

Heavy chain antibodies (HCAbs) of IgG2 and IgG3 subtypes were purified from the sera of Indian desert camel (Camelus dromedarius L.) by ammonium sulphate precipitation, followed by ion-exchange chromatography on DEAE-cellulose and affinity chromatography on protein A-sepharose and protein G-sepharose, and characterized by SDS-polyacrylamide gel electrophoresis, agar gel immunodiffusion (AGID), counter-immunoelectrophoresis (CIEP), immunoelectrophoresis (IEP), ELISA and immunoblotting. IgG2 and IgG3 were found to have molecular mass 46.77 kDa and 43.65 kDa, respectively by SDS-PAGE under reducing conditions. They migrated in beta-region in IEP and could be detected in CIEP, because of being more negatively charged and smaller size. Anti-camel IgG3 cross-reacted in AGID, ELISA and immunoblotting with IgGs of pig and ruminants (cattle, buffalo, sheep and goat), but not with immunoglobulins from horse, dog, guinea pigs, mice, fish, poultry and human. The present findings suggest close antigenic relationship of camels with pigs and ruminants.     

Analysis using a linear viscoelastic model of the in vitro osmotic kinetics of polydisperse synthetic colloids

This study clarifies the contribution to overall osmotic kinetics of colloid osmotic pressure (Pi) and the interaction of synthetic colloids with the membrane. Solutions (6%) of dextran with weight average molecular weight (MW(w)) 68 800 (DEX 70), dextran with MW(w) 40 000 (DEX 40), hydroxyethyl starch with MW(w) 70 000 (HES 70), gelatin with MW(w) 60 000 and albumin were tested. An osmotic flow cell fitted with membranes of molecular weight cutoff size 30 000 or 50 000 was used to measure time-dependent changes in Pi for each of these solutions. A linear viscoelastic model was fitted to the curve describing changes to Pi as a function of time. Values of total effective Pi for DEX 40 and DEX 70 were larger than those for HES 70, gelatin, and albumin. As an index of solute-solvent exchange rate at the membrane surface, these values were in the order DEX 40 > DEX 70, HES 70 > gelatin, albumin. The findings suggest that DEX 40 may be preferable for the temporary restoration of plasma volume because of a heightened initial osmotic force. In contrast, the osmotic force exerted by gelatin is slower to increase but is likely to be longer lasting in vivo as a result of the inhibition of gelatin from penetrating the capillary membrane due to its interaction with negatively charged groups in the endothelial glycocalyx.     

The pea chloroplast membrane-associated protein, IEP96, is a subunit of acetyl-CoA carboxylase

Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in pea ( Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and β-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterized and cloned. To further characterize the carboxyl-transferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidiaI-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the α-CT subunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the β-CT subunit. Furthermore, antibodies against β-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the β-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the α-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the α-CT subunit of pea chloroplast ACCase.     

Electrophoresis of colloidal biological particles

Microscope electrophoresis was used to measure the electrophoretic mobility of polystyrene latex particles and bacterial, and mammalian tissue cells. The submicroscopic hydrophilic colloids (gelatin, serum albumin, and staphylococcal enterotoxin B) were adsorbed on latex carrier particles to determine their electrophoretic mobility and the effect of concentration, pH, electrolyte addition, and buffer ionic strength. Mobility curves as a function of pH were established for latex particles at 1 ppm concentration indicating an isoelectric point (IEP) at pH 3.6. The IEP for Escherichia coli B cells was measured at pH 2.8, Serratia marcescens at pH 2.6, Bacillus subtilis var. niger at pH 2.9, and L strain mouse fibroblast cells at pH 4.4. Using an adsorption technique, isoelectric points were measured for proteins: gelatin (acid form) at pH 9.4, serum albumin at pH 4.9, and staphylococcal enterotoxin B at pH 6.3. Procefures for examining electrophoretic characteristics of microscopic and submicroscopic biological particles are described in order to standardize procedures and to generate results applicable to an understanding of parameters influencing concentration and purification of colloidal biological particles.     

Subunits of neurosteroid sulfatase from bovine brain

We have purified the neurosteroid sulfatase (NSS) from Triton X-100 solubilized microsomes of bovine brain about 100-fold. The purified enzyme is composed of two catalytic units (MW: 57 kDa) and two regulatory units (MW: 38 kDa), making it an alpha(2)beta(2) heterotetramer, whose apparent molecular weight was 180 kDa by gel filtration in the presence of Triton X-100.     

支架材料对棕色脂肪源性干细胞向起搏细胞诱导分化的影响

目的观察不同三维支架材料对棕色脂肪来源干细胞（BADSCs）诱导分化成起搏细胞的效果,为构建生物起搏器提供实验依据。 方法将培养7 d的原代BADSCs分别种植到胶原海绵、明胶海绵和透明质酸水凝胶3种不同的材料中,在不同时间用光镜和扫描电镜观察细胞-支架复合体中细胞形态学的变化,免疫荧光染色检测心肌细胞、起搏细胞相关蛋白的表达。采用单因素方差分析。 结果细胞在3种支架上均能存活、增殖,LIVE/DEAD检测显示,培养3 d的胶原海绵、明胶海绵和透明质酸水凝胶3种细胞-支架复合物死细胞率分别为（46.35±1.50）%、（47.00±1.60）%和（1.76±1.08）%,其中细胞在透明质酸水凝胶中死亡率最低,并且细胞-透明质酸水凝胶复合物可自发性地搏动,三组比较差异具有统计学意义（F = 37.56,P < 0.05）。培养至2周时,胶原海绵、明胶海绵和透明质酸水凝胶中Connexin45细胞阳性率分别为（10.67±1.25）%、（13.67±1.25）%和（21.00±1.60）%,差异有统计学意义（F = 9.435,P < 0.01）,HCN2细胞阳性率分别为（11.00±1.60）%、（14.00±2.16）%和（34.33±3.68）%,差异有统计学意义（F = 17.52,P < 0.01）,HCN4细胞阳性率分别为（18.67±2.05）%、（13.00±1.60）%和（66.00±2.94）%,差异有统计学意义（F = 27.96,P < 0.01）,Sr细胞阳性率分别为（13.00±1.63）%、（14.33±1.24）%和（75.33±3.30）%,差异有统计学意义（F = 36.40,P < 0.01）,水凝胶中Connexin45、HCN2、HCN4和Sr的细胞阳性率均高于胶原海绵和明胶海绵,差异均具有统计学意义（P < 0.05）。 结论BADSCs在胶原海绵、明胶海绵和透明质酸水凝胶中均能很好地生长和分化,但透明质酸水凝胶更适用于组织工程化起搏器的构建。     

Cytotoxicity and biocompatibility evaluation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery application

In this paper, covalently cross-linked hydrogel composed of N,O-carboxymethyl chitosan and oxidized alginate was developed intending for drug delivery application. In vitro/vivo cytocompatibility and biocompatibility of the developed hydrogel were preliminary evaluated. In vitro cytocompatibility test showed that the developed hydrogel exhibited good cytocompatibility against NH3T3 cells after 3-day incubation. According to the results of acute toxicity test, there was no obvious cytotoxicity for major organs during the period of 21-day intraperitoneal administration. Meanwhile, the developed hydrogel did not induce any cutaneous reaction within 72 h of subcutaneous injection followed by slow degradation and adsorption with the time evolution. Moreover, the extraction of developed hydrogel had nearly 0% of hemolysis ratio, which indicated the good hemocompatibility of hydrogel. Based on the above results, it may be concluded that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel with non-cytotoxicity and good biocompatibility might suitable for the various drug delivery applications.     

壳聚糖基角膜细胞载体的制备及其细胞相容性

为探讨羟丙基壳聚糖基共混膜作为组织工程技术中角膜细胞培养载体的可行性, 分别制备了羟丙基壳聚糖/硫酸软骨素、羟丙基壳聚糖/明胶/硫酸软骨素以及羟丙基壳聚糖/氧化透明质酸/硫酸软骨素三种共混膜。测定其透光率、含水量和蛋白吸附性能; 在共混膜上培养兔角膜上皮细胞, 通过观察角膜上皮细胞在不同载体膜上的生长状态、贴附情况, 测定细胞活性以及上清液中乳酸脱氢酶的活性, 研究三种壳聚糖基载体膜片与角膜上皮细胞的相容性。膜片理化性质测定结果表明三种共混膜片具有良好的透明度, 适宜的含水量和较强的蛋白吸附性能; 细胞相容性实验结果表明羟丙基壳聚糖/明胶/硫酸软骨素共混膜对细胞的损伤最小, 有利于细胞在膜上的贴附和生长, 表现出良好的细胞相容性, 有望作为角膜细胞载体体外构建组织工程化角膜。     

Gene delivery with low molecular weight linear polyethylenimines

BACKGROUND: Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation. METHODS: Polyplexes of plasmid DNA encoding for the enhanced green fluorescent protein (EGFP) and various LPEIs were compared concerning their transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells by flow cytometry. The involvement of endolysosomes in LPEI-mediated gene transfer was investigated by applying the proton pump inhibitor bafilomycin A1 and the lysosomotropic agent sucrose. Confocal laser scanning microscopy was applied to assess the size and shape of polyplexes under cell culture conditions, to detect their endolysosomal localization and to observe their translocation to the nucleus. RESULTS: The transfection efficiency could be altered by varying the MW and the amount of the polymer available for polyplex formation. The highest transfection efficiency (about 44%), i.e. the fraction of EGFP-positive cells, was obtained with LPEI 5.6 kDa, while the cytotoxicity remained low. The colocalization of polyplexes and endolysosomes was observed, and it appeared that the larger polyplexes escaped from the acidic organelles particularly quickly. For LPEI 5.0 and 9.0 kDa, the number of cells and nuclei that had taken up DNA after 6 hours was similar, as determined by flow cytometry. CONCLUSIONS: Our study suggests that LPEIs with low MWs are promising candidates for non-viral gene delivery, because they are more efficient and substantially less toxic than their higher MW counterparts.     

Mechanical Properties of Mammalian and Fish Gelatins as a Function of the Contents of α-Chain, β-Chain,and Low and High Molecular Weight Fractions

Small-strain oscillatory measurements and size-exclusion chromatography coupled to multiangle laser light scattering were used to study the mechanical properties and the molecular weight distribution, respectively, of acid porcine skin gelatins (type A), lime bovine bone gelatins (type B), and cold water fish gelatins, while principal component analysis (PCA) and partial least squares regression were used to relate the mechanical properties with the molecular weight distribution. The present study suggests a linear relationship between the mechanical properties and the fractions of low molecular weight (LMW) molecules, α-chains, β-chains, and high molecular weight (HMW) molecules. The Bloom value for mammalian gelatin was positively correlated with the fractions of α-chains, β-chains, and HMW molecules and negatively correlated with the fraction of LMW molecules. The dynamic storage modulus for cold water fish gelatin was positively correlated with the fractions of β-chains and HMW molecules and negatively correlated with the fractions of LMW molecules and α-chains.     

Probing Coagulation Behavior of Individual Aluminum Species for Removing Corresponding Disinfection Byproduct Precursors: The Role of Specific Ultraviolet Absorbance

Coagulation behavior of aluminum chloride and polyaluminum chloride (PACl) for removing corresponding disinfection byproduct (DBP) precursors was discussed in this paper. CHCl₃, bromine trihalomethanes (THM-Br), dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) formation potential yields were correlated with specific ultraviolet absorbance (SUVA) values in different molecular weight (MW) fractions of humic substances (HS), respectively. Correlation analyses and principal component analysis were performed to examine the relationships between SUVA and different DBP precursors. To acquire more structural characters of DBP precursors and aluminum speciation, freeze-dried precipitates were analyzed by fourier transform infrared (FTIR) and C 1s, Al 2p X-ray photoelectron spectroscopy (XPS). The results indicated that TCAA precursors (no MW limits), DCAA and CHCl₃ precursors in low MW fractions (MW<30 kDa) had a relatively good relations with SUVA values. These DBP precursors were coagulated more easily by in situ Al₁₃ of AlCl₃ at pH 5.0. Due to relatively low aromatic content and more aliphatic structures, THM-Br precursors (no MW limits) and CHCl₃ precursors in high MW fractions (MW>30 kDa) were preferentially removed by PACl coagulation with preformed Al₁₃ species at pH 5.0. Additionally, for DCAA precursors in high MW fractions (MW>30 kDa) with relatively low aromatic content and more carboxylic structures, the greatest removal occurred at pH 6.0 through PACl coagulation with aggregated Al₁₃ species.     

Physical and chemical properties of gelatin from the skin of cultured Amur sturgeon (Acipenser schrenckii)

To expand the usefulness of cultured Amur sturgeon, Acipenser schrenckii, its skin was used to explore the production of gelatin. After acetic acid pre‐treatment (0.05 m for 3 h), gelatin was extracted at temperatures of 50 or 70°C for 1 or 6 h. Gelatin yield ranged from 9.42 to 12.47% (wet weight basis) (P < 0.05). With increasing extraction time and temperature, the content of imino acids (proline + hydroxyproline), gel strength and L*‐value (lightness) decreased, while the a*‐value (redness) and b*‐value (yellowness) of gelatin gel increased (P < 0.05). Electrophoretic analysis revealed that α‐chains and β‐chains were predominant components in all extracted gelatins. Higher molecular weight proteins (γ‐chain) were also observed. Gelling and melting temperatures of gelatin were 13.6–14.6°C and 20.3–22.6°C, respectively. Circular dichroism (CD) spectra and Fourier transform infrared (FTIR) spectroscopy revealed the triple helix loss in gelatin (A₁₂₃₅ (AIII)/A₁₄₅₁ < 1). Extraction conditions caused secondary structure changes in the gelatin. More likely due to the differences in the culture water temperature, gelatin exhibited gelling and melting temperatures intermediate between those of cold‐ and warm‐water fish gelatins. The obtained gelatin can be used in food products or in the production of bioactive compounds.     

Rapidly photo-cross-linkable chitosan hydrogel for peripheral neurosurgeries

Restoring continuity to severed peripheral nerves is crucial to regeneration and enables functional recovery. However, the two most common agents for coaptation, sutures and fibrin glues, have drawbacks such as inflammation, pathogenesis, and dehiscence. Chitosan-based adhesives are a promising alternative, reported to have good cytocompatibility and favorable immunogenicity. A photo-cross-linkable hydrogel based on chitosan is proposed as a new adhesive for peripheral nerve anastomosis. Two Az-chitosans were synthesized by conjugating 4-azidobenzoic acid with low (LMW, 15 kDa) and high (HMW, 50-190 kDa) molecular weight chitosans. These solutions formed a hydrogel in less than 1 min under UV light. The LMW Az-chitosan was more tightly cross-linked than the HMW variant, undergoing significantly less swelling and possessing a higher rheological storage modulus, and both Az-chitosan gels were stiffer than commercial fibrin glue. Severed nerves repaired by Az-chitosan adhesives tolerated longitudinal forces comparable or superior to fibrin glue. Adhesive exposure to intact nerves and neural cell culture showed both Az-chitosans to be nontoxic in the acute (minutes) and chronic (days) time frames. These results demonstrate that Az-chitosan hydrogels are cytocompatible and mechanically suitable for use as bioadhesives in peripheral neurosurgeries.     

Interaction of heme proteins with poly(propyleneimine) dendrimers in layer-by-layer assembly films under different pH conditions

With the isoelectric point at pH 7.4, hemoglobin (Hb) has net positive surface charges at pH 5.0 and overall negative charges at pH 9.0, and is essentially neutral at pH 7.0. The fifth-generation poly(propyleneimine) (PPI) dendrimer is usually positively charged in aqueous solution. The {PPI/Hb}n films under different pH conditions have been successfully fabricated on various solid surfaces by the layer-by-layer assembly technique, and the growth of films was monitored by ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV). Not only was the negatively charged Hb at pH 9.0 alternately adsorbed with positively charged PPI onto solid substrates by electrostatic attraction between them, but the positively charged Hb at pH 5.0 was also successfully assembled with like charged PPI into layer-by-layer {PPI/Hb(pH 5.0)}n films. For the latter, the localized electrostatic interaction or the charge reversal of proteins on PPI surface may be the main driving force. For {PPI/Hb(pH 7.0)}n films, however, the hydrophobic/hydrophilic interaction may play a more important role in the assembly, making the amount of adsorbed Hb even less than that of {PPI/Hb(pH 5.0)}n films. For comparison, negatively charged catalase (Cat) at pH 8.0 was used to assemble layer-by-layer films with positive PPI, but {PPI/Cat}n films showed quite different properties from {PPI/Hb}n films. UV-vis and infrared (IR) spectroscopy, QCM, ellipsometry, and voltammetry were utilized to characterize the {PPI/protein}n films. The results suggest that the proteins in the multilayer films retain their near-native structure and display good voltammetric response for heme Fe(III)/Fe(II) redox couples at underlying pyrolytic graphite (PG) electrodes. Electrocatalysis of oxygen and hydrogen peroxide based on direct electrochemistry of heme proteins at {PPI/protein}n film electrodes was also demonstrated.