A Double-Needle Gold-Silver Electrodes Continuous Glucose Monitoring Device

ObjectivesDiabetes is a serious, long-term disease and the use of continuous glucose monitoring sensors can reduce reliance on other painful invasive blood testing methods such as the finger blood glucose test. According to our work, a low-cost continuous glucose sensor has been developed based on electrochemical measurement techniques.MaterialsThe sensor is based on a two needles system; a gold and a silver electrode are integrated into a circular shaped electronic printed circuit board (PCB). The sensing part is based on biological electrochemical measurements. Glucose oxidase (Gox) was used as the active sensing element and ferrocene (Fc) as a mediator. Simple and low-cost coating methods were used; these methods are self-assembled monolayers and deep coating. This will reduce the final cost of the sensor as no expensive technique was used. The electrical subsystem contains a low-noise and low-power trans-impedance front-end as well as a single-chip low-power Bluetooth microcontroller with a 12-bit Analog-to-Digital Converter (ADC).ResultsThe sensor was tested in various concentrations of glucose. As a result of initial in vitro experiments, detailed analytical performance metrics are presented. The device has consistently shown a sensitivity of 3.059 mV/(mg/dl) reading with a linear range of 0-400 mg/dl.ConclusionThe proposed study shows promising results for glucose detection. Thus, this type of sensor can be used for different analyzes targeting biological applications after further investigations and analysis.     

A genomic search approach to identify carbonyl reductases in Gluconobacter oxydans for enantioselective reduction of ketones

The versatile carbonyl reductases from Gluconobacter oxydans in the enantioselective reduction of ketones to the corresponding alcohols were exploited by genome search approach. All purified enzymes showed activities toward the tested ketoesters with different activities. In the reduction of 4-phenyl-2-butanone with in situ NAD(P)H regeneration system, (S)-alcohol was obtained with an e.e. of up to 100% catalyzed by Gox0644. Under the same experimental condition, all enzymes catalyzed ethyl 4-chloroacetoacetate to give chiral products with an excellent e.e. of up to 99%, except Gox0644. Gox2036 had a strict requirement for NADH as the cofactor and showed excellent enantiospecificity in the synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate. For the reduction of ethyl 2-oxo-4-phenylbutyrate, excellent e.e. (>99%) and high conversion (93.1%) were obtained by Gox0525, whereas the other enzymes showed relatively lower e.e. and conversions. Among them, Gox2036 and Gox0525 showed potentials in the synthesis of chiral alcohols as useful biocatalysts.     

Vinyl ketone reduction by three distinct Gluconobacter oxydans 621H enzymes

Three cytosolic NADPH-dependent flavin-associated proteins (Gox2107, Gox0502, and Gox2684) from Gluconobacter oxydans 621H were overproduced in Escherichia coli, and the recombinant enzymes were purified and characterized. Apparent native molecular masses of 65.2, 78.2, and 78.4 kDa were observed for Gox2107, Gox0502, and Gox2684, corresponding to a trimeric structure for Gox2107 and dimers for Gox0502 and Gox2684. Analysis of flavin content revealed Gox2107 was flavin adenine dinucleotide dependent, whereas Gox0502 and Gox2684 contained flavin mononucleotide. The enzymes were able to reduce vinyl ketones and quinones, reducing the olefinic bond of vinyl ketones as shown by ¹H nuclear magnetic resonance. Additionally, Gox0502 and Gox2684 stereospecifically reduced 5S-(+)-carvone to 2R,5S-dihydrocarvone. All enzymes displayed highest activities with 3-butene-2-one and 1,4-naphthoquinone. Gox0502 and Gox2684 displayed a broader substrate spectrum also reducing short-chain α-diketones, whereas Gox2107 was most catalytically efficient.     

Computational design of short-chain dehydrogenase Gox2181 for altered coenzyme specificity

Short-chain dehydrogenase Gox2181 from Gluconobacter oxydans catalyzes the reduction of 2,3-pentanedione by using NADH as the physiological electron donor. To realize its synthetic biological application for coenzyme recycling use, computational design and site-directed mutagenesis have been used to engineer Gox2181 to utilize not only NADH but also NADPH as the electron donor. Single and double mutations at residues Q20 and D43 were made in a recombinant expression system that corresponded to Gox2181-D43Q and Gox2181-Q20R&D43Q, respectively. The design of mutant Q20R not only resolved the hydrogen bond interaction and electrostatic interaction between R and 2′-phosphate of NADPH, but also could enhance the binding with 2′-phophated of NADPH by combining with D43Q. Molecular dynamics simulation has been carried out to testify the hydrogen bond interactions between mutation sites and 2′-phosphate of NADPH. Steady-state turnover measurement results indicated that Gox2181-D43Q could use both NADH and NADPH as its coenzyme, and so could Gox2181-Q20R&D43Q. Meanwhile, compared to the wild-type enzyme, Gox2181-D43Q exhibited dramatically reduced enzymatic activity while Gox2181-Q20R&D43Q successfully retained the majority of enzymatic activity.     

Electroless-plated gold films for sensitive surface plasmon resonance detection of white spot syndrome virus

The paper describes the rapid and label-free detection of the white spot syndrome virus (WSSV) using a surface plasmon resonance (SPR) device based on gold films prepared by electroless plating. The plating condition for obtaining films suitable for SPR measurements was optimized. Gold nanoparticles adsorbed on glass slides were characterized by transmission electron microscopy (TEM). Detection of the WSSV was performed through the binding between WSSV in solution and the anti-WSSV single chain variable fragment (scFv antibody) preimmobilized onto the sensor surface. Morphologies of the as-prepared gold films, gold films modified with self-assembled alkanethiol monolayers, and films covered with antibody were examined using an atomic force microscope (AFM). To demonstrate the viability of the method for real sample analysis, WSSV of different concentrations present in a shrimp hemolymph matrix was determined upon optimizing the surface density of the antibody molecules. The SPR device based on the electroless-plated gold films is capable of detecting concentration of WSSV as low as 2.5 ng/mL in 2% shrimp hemolymph, which is one to two orders of magnitude lower than the level measurable by enzyme-linked immunosorbant assays.     

Characterization of a Putative Stereoselective Oxidoreductase from Gluconobacter oxydans and Its Application in Producing Ethyl (R)-4-Chloro-3-Hydroxybutanoate Ester

A gene encoding an NADH-dependent short-chain dehydrogenase/reductase (gox2036) from Gluconobacter oxydans 621H was cloned and heterogeneously expressed in Escherichia coli. The protein (Gox2036) was purified to homogeneity and biochemically characterized. Gox2036 was a homotetramer with a subunit size of approximately 28 kDa. Gox2036 had a strict requirement for NAD⁺/NADH as the cofactor. Gox2036 displayed preference for oxidation of secondary alcohols and 2,3-diols as well as for reduction of α-diketones, hydroxy ketones, α-ketoesters, and β-ketoesters. However, Gox2036 was poorly active on 1,2-diols and acetoin and showed no activity on primary alcohols, polyols, and aldehydes. The optimum pH values for the oxidation and reduction reactions were 9 and 6, respectively. Gox2036 was highly selective in the reduction of various β-ketones and β-ketoesters. Among the substrates tested, ethyl 4-chloro acetoacetate was reduced to ethyl (R)-4-chloro-3-hydroxybutanoate ester with an excellent conversion yield of 96.9 % and optical purity of >99 % e.e. using an efficient in situ NADH-recycling system involving glucose and a glucose dehydrogenase from Bacillus subtilis (BsGDH).     

Glucose sensors made of novel carbon nanotube-gold nanoparticle composites

Carbon nanotube and metal particle composites have been exploited to fabricate high performance electrochemical devices. However, the physical and chemical procedures to synthesize the composites are labor intensive and inefficient. Our study reveals an one-step wet chemistry method to accomplish fast and controllable production of gold nanoparticle (AuNP) and carbon naotube (CNT) composites. Such a process is sensitive to the surface charge. Especially, when functionalized with carboxyl groups, the CNTs carried negative charges and showed low level association with negatively charged AuNPs. Thermal treatment was employed to decompose the carboxyl groups and render each CNT a charge-free surface thereby achieving a high level AuNP-CNT association. The fabricated glucose sensors demonstrated dependence of their sensitivities to the amount of AuNPs on the CNTs. The enhancement of sensitivity can be attributed to an accelerated electron transfer rate from glucose oxidase Gox to the electrode. The Michaelis-Menten kinetics also indicated improved performance in the glucose sensor made of AuNP-CNTs. Therefore, our research revealed a novel approach to produce metallic nanoparticle and CNT composite for fabricating high performance electrochemical sensors.     

Postnatal ontogeny of glucose homeostasis and insulin action in sheep

Glucose tolerance declines with maturation and aging in several species, but the time of onset and extent of changes in insulin sensitivity and insulin secretion and their contribution to changes in glucose tolerance are unclear. We therefore determined the effect of maturation on glucose tolerance, insulin secretion, and insulin sensitivity in a longitudinal study of male and female sheep from preweaning to adulthood, and whether these measures were related across age. Glucose tolerance was assessed by intravenous glucose tolerance test (IVGTT, 0.25 g glucose/kg), insulin secretion as the integrated insulin concentration during IVGTT, and insulin sensitivity by hyperinsulinemic-euglycemic clamp (2 mU insulin.kg(-1).min(-1)). Glucose tolerance, relative insulin secretion, and insulin sensitivity each decreased with age (P < 0.001). The disposition index, the product of insulin sensitivity, and various measures of insulin secretion during fasting or IVGTT also decreased with age (P < 0.001). Glucose tolerance in young adult sheep was independently predicted by insulin sensitivity (P = 0.012) and by insulin secretion relative to integrated glucose during IVGTT (P = 0.005). Relative insulin secretion before weaning was correlated positively with that in the adult (P = 0.023), whereas glucose tolerance, insulin sensitivity, and disposition indexes in the adult did not correlate with those at earlier ages. We conclude that glucose tolerance declines between the first month of life and early adulthood in the sheep, reflecting decreasing insulin sensitivity and absence of compensatory insulin secretion. Nevertheless, the capacity for insulin secretion in the adult reflects that early in life, suggesting that it is determined genetically or by persistent influences of the perinatal environment.     

Bienzyme HRP-GOx-modified gold nanoelectrodes for the sensitive amperometric detection of glucose at low overpotentials

Gold nanotubular electrode ensembles were prepared by using electroless deposition of the metal within the pores of polycarbonate track-etched membranes. Mono-enzyme (GOx) and monolayer/bilayer bienzyme (GOx/HRP) bioelectrodes were prepared by immobilizing the enzymes onto gold nanotubes surfaces modified with mercaptoethylamine. Batch amperometric responses to glucose for the different bioelectrodes were determined and compared. The response of the two geometries (monolayer and bilayer) of the bienzyme electrodes was shown to vary with regard to sensitivity at detection potentials above 0V. On the contrary, at detection potentials below 0V, no noticeable influence of the configuration of the bienzyme on the response intensity was observed. The mono-enzyme (650 microAmM-1 in benzoquinone (BQ) at -0.8 V versus Ag/AgCl) and the two bienzyme bioelectrodes (+/-400 microAmM-1 in hydroquinone (H2Q) at -0.2V versus Ag/AgCl) display remarkable sensitivities compared to a classical GOx-modified gold macroelectrode (13 microAmM-1 in BQ at -0.8 V versus Ag/AgCl). A remarkable feature of the bienzyme electrodes is the possibility to detect glucose at very low applied potentials where the noise level and interferences from other electro-oxidizable compounds are minimal. Another important characteristic of the monolayer bienzyme electrode is the possible existence of a direct electronic communication between HRP and the transducer surface.     

ACCELERATION OF YOGURT FERMENTATION BY BACTERIAL MEMBRANE FRACTION BIOCATALYSTS

Bacterial membrane fraction biocatalysts obtained from Escherichia coli (E-8), Gluconobacter oxydans (Gox) and Acetobacter xylinum (Acx), as well as the commercial oxygen scavenger Oxyrase^TM, at concentrations of 0.1–2.0 U/ml effectively stimulated cell growth of Lactobacillus bulgaricus and Sreptococcus thermophilus, and lactic acid production during yogurt fermentation. The membrane fractions scavenged oxygen in the fermented milk to an optimal oxygen tension for growth of yogurt cultures. The yogurt culture populations with membrane fraction(s) increased faster than those without. Total counts in the presence of yogurts with Oxyrase, E-8, Gox, and Acx were 0.5–1, 0.5, 1.5, or 1.2 log cycles, respectively, greater than counts of the control after 3 h of fermentation. The commercial membrane fraction Oxyrase reduced the fermentation time by 1 h needed to reach pH 4.5 compared to the controls, while E-8, Gox, and Acx reduced time by about 0.5 h, 1.5 h and 1–1.5 h, respectively, depending on the membrane concentrations. The titratable acidity was corresponded well with the reduction in pH. Ratios of rods to cocci among the samples with and without membrane fraction supplementation were not different (P > .05). Each membrane fraction biocatalyst enhanced the depletion rate of dissolved oxygen in a yogurt mix differently.     

Glucose uptake after resistance training of different intensities but of equal work volume

High-intensity (HI) resistance exercise augments postexercise glucose uptake to a greater degree than low-intensity (LO) resistance exercise; however, few studies have equated the work volumes between intensity levels. The purpose of this study was to compare the effect of acute HI and LO resistance exercise of equal work volume on glucose uptake in resistant-trained men. Fifteen healthy male (22.9 +/- 3.8 years old), resistance-trained (6.7 +/- 3.9 years) subjects completed three treatment sessions: CON (no-exercise control), HI (3 x 8, 85% 10-RM), and LO (3 x 15, 45% 10-RM). HI and LO sessions consisted of eight exercises. Glucose uptake was measured the following morning by using the hyperinsulinemic euglycemic clamp technique. Glucose disposal was measured by analyzing the glucose infusion rate during the final 30 minutes of steady-state blood glucose concentrations. Insulin sensitivity was calculated by dividing the glucose infusion rate by the average insulin infusion. Results indicate that fasting blood glucose levels were not significantly different among treatment sessions (CON = 80.5 +/- 5.3 versus HI = 77.0 +/- 4.9 versus LO = 77.1 +/- 6.0 mg.dL). Glucose uptake was not significantly different among treatment sessions (CON = 11.3 +/- 3.0 versus HI = 11.7 +/- 2.7 versus LO = 11.4 +/- 2.8 mg.kg FFM.min). Insulin sensitivity did not change among treatment sessions (CON = 0.26 +/- 0.09 versus HI = 0.28 +/- 0.07 versus LO = 0.27 +/- 0.06 (mg.kg FFM.min)/(uU.mL)). The data indicate that the resistance training sessions did not modify acute insulin sensitivity. This may have been because of the high levels of fitness of the subjects, which allowed for the cellular adaptations for enhanced insulin sensitivity and glucose uptake that are unaffected by this volume of acute exercise.     

A common precursor for the three subunits of L-glutamate oxidase encoded by gox gene from Streptomyces platensis NTU3304

A segment of DNA containing the L-glutamate oxidase (gox) gene from Streptomyces platensis NTU3304 was cloned. The entire nucleotide sequence of the protein-coding portion consisting of 2130 bp (710 codons, including AUG and UGA) of the cloned DNA fragment was determined. The gox gene contained only one open reading frame (ORF) which coded for a 78-kDa polypeptide, the precursor of active extracellular Gox. Mature Gox is composed of three subunits, designated as alpha, beta, and gamma, with molecular masses of 39, 19, and 16 kDa, respectively. Analyses of the N-terminal amino acid sequences of the subunits revealed that the order of subunits in the precursor polypeptide encoded by the ORF, from N-terminus to C-terminus, is alpha-gamma-beta. The presence of the flavin adenine dinucleotide (FAD)-binding motif place Gox as a member of the flavoenzyme family. Furthermore, a negative effect of glucose on the biosynthesis of Gox was observed when it was used as carbon source.     

Analysis of aldehyde reductases from Gluconobacter oxydans 621H

Two cytosolic nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductases, Gox1899 and Gox2253, from Gluconobacter oxydans 621H were overproduced and purified from Escherichia coli. The purified proteins exhibited subunit masses of 26.4 (Gox1899) and 36.7 kDa (Gox2253). Both proteins formed homo-octamers exhibiting native masses of 210 and 280 kDa, respectively. The substrate spectra, optimal reaction conditions, and kinetic constants were determined for Gox1899 and Gox2253. Both enzymes efficiently catalyzed the reduction of medium/long-chain aldehydes. However, Gox1899 had a wider substrate spectrum and was more catalytically efficient. The best activity with Gox1899 was found for aliphatic aldehydes of C6-C10. In contrast, Gox2253 had a limited substrate spectrum and reduced octanal, nonanal, and decanal. Both enzymes were unable to oxidize primary alcohols. Aldehyde removal may be of particular importance for Gluconobacter because the membrane-bound alcohol dehydrogenase rapidly oxidizes short to long-chain alcohols, and large quantities of aldehydes could enter the cell, making detoxification necessary.     

A comparison of different strategies for the construction of amperometric enzyme biosensors using gold nanoparticle-modified electrodes

A comparison of the analytical performances of several enzyme biosensor designs, based on the use of different tailored gold nanoparticle-modified electrode surfaces, is discussed. Glucose oxidase (GOx) and the redox mediator tetrathiafulvalene were coimmobilized in all cases by crosslinking with glutaraldehyde. The biosensor designs tested were based on the use of (i) colloidal gold (Au(coll)) bound on cysteamine (Cyst) monolayers self-assembled on a gold disk electrode (AuE) and (ii) glassy carbon electrodes (GCEs) modified with electrodeposited gold nanoparticles (nAu). The results obtained with these designs were compared with those provided by a GOx/Cyst-AuE and a GOx/MPA-AuE. In the second case (ii), configurations based on direct immobilization of GOx on nAu (GOx/nAu-GCE) or on Cyst or MPA self-assembled monolayers (SAMs) previously bound on gold nanoparticles (GOx/Cyst-nAu-GCE or GOx/MPA-nAu-GCE, respectively) were compared. The analytical characteristics of glucose calibration plots and the kinetic parameters of the enzyme reaction were compared for all of the biosensors tested. The GOx/Au(coll)-Cyst-AuE design showed a sensitivity for glucose determination higher than that achieved with GOx/Cyst-AuE and GOx/Au(coll)-Cyst/Cyst-AuE and similar to that achieved with GOx/MPA-AuE. Moreover, the useful lifetime of one single GOx/Au(coll)-Cyst-AuE was 28 days, remarkably longer than that of the other GOx biosensor designs.     

Characterization of two aldo–keto reductases from Gluconobacter oxydans 621H capable of regio- and stereoselective α-ketocarbonyl reduction

Two cytosolic NADPH-dependent carbonyl reductases from Gluconobacter oxydans 621H, Gox0644 and Gox1615, were heterologously produced in Escherichia coli. The recombinant proteins were purified to homogeneity and characterized. Gox0644 and Gox1615 were dimers with native molecular masses of 66.1 and 74.5 kDa, respectively. The enzymes displayed broad substrate specificities and reduced α-ketocarbonyls at the keto moiety most proximal to the terminus of the alkyl chain to produce alpha-hydroxy carbonyls, as demonstrated by NMR. With respect to stereoselectivity, protein Gox0644 specifically reduced 2,3-pentanedione to 2R-hydroxy-pentane-3-one, whereas Gox1615 produced 2S-hydroxy-pentane-3-one. Both enzymes also reduced 1-phenyl-1,2-propanedione to 2-hydroxy-1-phenylpropane-1-one, which is a key intermediate in the production of numerous pharmaceuticals, such as antifungal azoles and antidepressants. Gox0644 displayed highest activities with 2,3-diones, α-ketoaldehydes, α-keto esters, and 2,5-diketogluconate. Gox1615 was less active with these substrates, but displayed a broader substrate spectrum reducing a variety of α-diketones and aldehydes.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy

Summary One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

Biochemical and structural analysis of Gox2181, a new member of the SDR superfamily from Gluconobacter oxydans

Gluconobacter oxydans enable to oxidize sugars and polyols incompletely to corresponding materials with potential industrial applications, containing around 75 putative dehydrogenases. One of these putative dehydrogenases, Gox2181, was cloned and expressed in Escherichia coli BL21 (DE3), and its X-ray crystal structure was determined to a resolution of 1.8 Å. Gox2181 formed a homo-tetramer in the crystal that was coincident with the apparent molecular mass determined in the solution. Gox2181 displayed α/β-folding patterns, the conserved catalytic tetrad of Asn119-Ser147-Tyr162-Lys166, and the NAD-binding pocket, which aligned well with the ‘classical’ type of short-chain dehydrogenase/reductase (SDR) enzymes. Gox2181 was denoted SDR51C based on the SDR nomenclature system. The purified recombinant Gox2181 was demonstrated to be NAD(H)-dependent and active towards a wide range of substrates, including sugar alcohols, secondary alcohols, ketones, and ketoses. Among the substrates tested, Gox2181 displayed preference for secondary hydroxyl or carbonyl groups, showing low K_m values with d-arabitol and butanedione.     

Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium that lacks a complete Embden–Meyerhof pathway (glycolysis). The organism instead uses the pentose phosphate pathway to oxidize sugars and their phosphorylated intermediates. However, the lack of glycolysis limits the amount of NADH as electron donor for electron transport phosphorylation. It has been suggested that the pentose phosphate pathway contributes to NADH production. Six enzymes predicted to play central roles in intracellular glucose and gluconate flux were heterologously overproduced in Escherichia coli and characterized to investigate the intracellular flow of glucose and gluconates into the pentose phosphate pathway and to explore the contribution of the pentose phosphate pathway to NADH generation. The key pentose phosphate enzymes glucose 6-phosphate dehydrogenase (Gox0145) and 6-phosphogluconate dehydrogenase (Gox1705) had dual cofactor specificities but were physiologically NADP- and NAD-dependent, respectively. Putative glucose dehydrogenase (Gox2015) was NADP-dependent and exhibited a preference for mannose over glucose, whereas a 2-ketogluconate reductase (Gox0417) displayed dual cofactor specificity for NAD(P)H. Furthermore, a putative gluconokinase and a putative glucokinase were identified. The gluconokinase displayed high activities with gluconate and is thought to shuttle intracellular gluconate into the pentose phosphate pathway. A model for the trafficking of glucose and gluconates into the pentose phosphate pathway and its role in NADH generation is presented. The role of NADPH in chemiosmotic energy conservation is also discussed.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

Energetic studies of lactose active transport in Escherichia coli membrane vesicles

The energetics of D-lactate-driven active transport of lactose in right-side-out Escherichia coli membrane vesicles has been investigated with a microcalorimetric method. Changes of enthalpy (delta Hox), free energy (delta Gox), and entropy (delta Sox) during the D-lactate oxidation reaction in the presence of membrane vesicles are -39.9 kcal, -46.4 kcal, and 22 cal/deg per mole of D-lactate, respectively. The free energy released by this reaction is utilized to form a proton electrochemical potential (delta-microH+) across the membrane. The higher observed heat in the D-lactate oxidation reaction in the presence of carbonylcyanide m-chlorophenylhydrazone (a proton ionophore) supports the postulate that delta-microH+ is formed across the membrane vesicles. Thermodynamic quantities for the formation of delta-microH+ are delta Hm = 14.1 kcal, delta Gm = 0.6 kcal, and delta Sm = 45 cal/deg per mole of D-lactate. The efficiency in the free energy transfer from the oxidation reaction to the formation of delta-microH+ (defined by delta Gm/delta Gox) was 2%, as compared to that in the heat transfer (defined by delta Hm/delta Hox) of 35%. The energetics of the movement of lactose in symport with proton across the membrane as a consequence of the formation of delta-microH+ are delta H1 = -19 kcal, delta G1 = -0.5 kcal, and delta S1 = -62 cal/deg per mole of lactose. No heat of reaction is contributed by lactose movement across the membrane without symport with H+.