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1.
Myoglobin (Mb) and its iron have been proposed to be major prooxidants in cooked meats. To understand the mechanisms and differentiate between the prooxidant and antioxidant potential of oxymyoglobin (OxyMb) and metmyoglobin (MetMb), their prooxidant activity, iron content, solubility, free radical scavenging activity, and iron binding capacity were determined as a function of thermal processing. The ability of native and heat denatured OxyMb and MetMb to promote the oxidation of muscle microsomes was different. MetMb promoted lipid oxidation in both its native and denatured states. Conversely, OxyMb became antioxidative when the protein was heated to temperatures >or=75 degrees C. The increased antioxidant activity of heat denatured OxyMb was likely due to a decrease in its prooxidative activity due to its loss of solubility. These data show that the impact on oxidative reactions of Mb is the result of the balance between its antioxidant and prooxidant activities.  相似文献   

2.
alpha,beta-unsaturated aldehydes accelerate oxymyoglobin oxidation.   总被引：3，自引：0，他引：3  
This study investigates the potential basis for enhancement of oxymyoglobin (OxyMb) oxidation by lipid oxidation products. Aldehydes known to be formed as secondary lipid oxidation products were combined with OxyMb in aqueous solution at 37 degrees C and pH 7.4. Metmyoglobin (MetMb) formation was greater in the presence of alpha,beta-unsaturated aldehydes than their saturated counterparts of equivalent carbon chain length. Additionally, increasing chain length from hexenal through nonenal resulted in increased MetMb formation (P < 0.05). Electrospray ionization mass spectrometry (ESI-MS) revealed that OxyMb incubated with 4-hydroxynonenal (HNE) at pH 7.4 at 37 degrees C yielded myoglobin molecules adducted with one to three molecules of HNE from 0.5 to 2 h of incubation, respectively. A prooxidant effect of HNE was noted at pH 7.4 but was not apparent at pH 5.6 when compared to the control (P < 0.05). This appeared to be due to rapid OxyMb autoxidation at this pH compared to pH 7.4. ESI-MS demonstrated that adduction of HNE to OxyMb occurred at pH 5.6. This research demonstrates that alpha, beta-unsaturated aldehydes accelerate OxyMb oxidation and appear to do so via covalent attachment.  相似文献   

3.
The interaction between fish myoglobin (Mb) and natural actomyosin (NAM) extracted from fresh and frozen fish was studied. The quantity of soluble Mb in Mb-NAM extracted was less in frozen than in fresh fish (P < 0.05). However, no differences were observed in Mb that remained in solution following preparation of Mb-NAM from frozen whole fish vs frozen fillets (P > 0.05). MetMb formation in Mb-NAM was generally greater than that observed in control Mb (P < 0.05); the greatest MetMb content occurred in Mb-NAM extracted from frozen whole fish (P < 0.05). The effect of different aldehyde oxidation products on the interaction between fish Mb and NAM was also studied in vitro. The loss of soluble Mb from NAM:Mb preparations was greater in the presence of hexenal and hexanal (P < 0.05) relative to controls, and the degree of solubility loss varied with aldehyde type. Hexenal caused greater OxyMb oxidation than hexanal (P < 0.05). Whiteness of washed NAM and NAM-Mb mixtures decreased following aldehyde addition (P < 0.05). In the absence of Mb, the Ca2+ -ATPase activity of NAM was lower with added hexenal than with hexanal (P < 0.05). However, no differences in Ca2+ -ATPase activity between hexanal and hexenal-treated samples were observed when Mb was present (P > 0.05). Reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses suggested that both disulfide and nondisulfide covalent linkages contributed to aldehyde-induced cross-linking between Mb and myofibrillar proteins.  相似文献   

4.
Effect of glutathione on oxymyoglobin oxidation   总被引：4，自引：0，他引：4  
The oxidation of oxymyoglobin (OxyMb) to metmyoglobin (MetMb) is responsible for fresh meat discoloration. Glutathione (GSH) is an important tripeptide reductant that can protect lipid and protein from oxidation. The objective of this research was to investigate the effect of GSH on MetMb formation in vitro and in bovine skeletal muscle cytosol. Equine MetMb formation was greater in the presence of GSH than controls at pH 5.6 or 7.2 and 25 or 37 degrees C (p < 0.05); GSH addition to purified bovine OxyMb solution also resulted in more MetMb formation at pH 7.2 and 25 or 37 degrees C (p < 0.05). This effect on MetMb formation was partly or completely inhibited by EDTA or catalase in the GSH-equine OxyMb system (p < 0.05). The addition of GSH to bovine muscle cytosol inhibited MetMb formation at pH 5.6 or 7.2 and 4 or 25 degrees C (p < 0.05); the effect was concentration-dependent. The inhibitory effect was observed in a high molecular weight (HMW) but not low molecular weight fraction of cytosol at pH 7.2 and 25 degrees C (p < 0.05); there was no effect when HMW was heated at 90 degrees C for 15 min. These results suggest the antioxidant effect of GSH on bovine OxyMb is dependent on heat-sensitive HMW cytosolic component(s).  相似文献   

5.
The objective of this study was to assess the morphological integrity and functional potential of mitochondria from postmortem bovine cardiac muscle and evaluate mitochondrial interactions with myoglobin (Mb) in vitro. Electron microscopy revealed that mitochondria maintained structural integrity at 2 h postmortem; prolonged storage resulted in swelling and breakage. At 2 h, 96 h, and 60 days postmortem, the mitochondrial state III oxygen consumption rate (OCR) and respiratory control ratio decreased with time at pH 7.2 and 5.6 (p < 0.05). Mitochondria isolated at 60 days did not exhibit ADP-induced transitions from state IV to state III oxygen consumption. Tissue oxygen consumption also decreased with time postmortem (p < 0.05). Mitochondrial oxygen consumption was inhibited by decreased pH in vitro (p < 0.05). In a closed system, mitochondrial respiration resulted in decreased oxygen partial pressure (pO(2)) and enhanced conversion of oxymyoglobin (OxyMb) to deoxymyoglobin (DeoMb) or metmyoglobin (MetMb). Greater mitochondrial densities caused rapid decreases in pO(2) and favored DeoMb formation at pH 7.2 in closed systems (p < 0.05); there was no effect on MetMb formation (p > 0.05). MetMb formation was inversely proportional to mitochondrial density at pH 5.6 in closed systems. Mitochondrial respiration in open systems resulted in greater MetMb and DeoMb formation at pH 5.6 and pH 7.2, respectively, vs controls (p < 0.05). The greatest MetMb formation was observed with a mitochondrial density of 0.5 mg/mL at both pH values in open systems. Mitochondrial respiration facilitated a shift in Mb form from OxyMb to DeoMb or MetMb, and this was dependent on pH, oxygen availability, and mitochondrial density.  相似文献   

6.
We evaluated the usefulness of the ratio A503/A581 as a browning index (BI) for estimating brown color formation in solutions containing oxymyoglobin (OxyMb) and carboxymyoglobin (COMb). In split-chamber cuvette analyses with different proportions of metmyoglobin (MetMb), COMb and OxyMb, BI was highly correlated (r = 0.93-0.94) with direct estimation of MetMb. Moreover, A503/A581 was not influenced by different COMb-OxyMb proportions. Second, we investigated 4-hydroxy-2-nonenal (HNE)-induced spectral changes in OxyMb and COMb solutions. At pH 7.4 and 37 degrees C, BI was greater in HNE-treated OxyMb and COMb samples than in aldehyde-free controls (P < 0.05). However, at pH 5.6 and 4 degrees C, HNE-induced browning was more pronounced in COMb than in OxyMb. These results indicated that COMb is susceptible to lipid-oxidation-induced browning in a pH- and temperature-dependent manner.  相似文献   

7.
Off-flavor and discoloration of meat products result from lipid oxidation and myoglobin (Mb) oxidation, respectively, and these two processes appear to be interrelated. The objective of this study was to investigate their potential interaction in mitochondria and the effects of mitochondrial alpha-tocopherol concentrations on lipid oxidation and metmyoglobin (MetMb) formation in vitro. The addition of ascorbic acid and ferric chloride (AA-Fe(3+)) increased ovine and bovine mitochondrial lipid oxidation when compared with their controls (p < 0.05); MetMb formation also increased with increased lipid oxidation relative to controls (p < 0.05). Reactions containing Mb and mitochondria with greater alpha-tocopherol concentrations demonstrated less lipid oxidation and MetMb formation than mitochondria with lower alpha-tocopherol concentrations. Greater mitochondrial alpha-tocopherol concentration was also correlated with increased mitochondrial oxygen consumption in vitro and with a more pronounced effect at pH 7.2 than at pH 5.6. Relative to controls, succinate addition to bovine mitochondria resulted in increased concentrations of ubiquinol 10 and alpha-tocopherol and decreased lipid and Mb oxidation (p < 0.05). Mitochondrial lipid oxidation was closely related to MetMb formation; both processes were inhibited by alpha-tocopherol in a concentration-dependent manner.  相似文献   

8.
The effect of the lipid oxidation product, 4-hydroxy-2-nonenal (HNE), on oxidation of oxymyoglobin (OxyMb) from seven different meat-producing species was investigated. Relative to controls, HNE increased OxyMb oxidation within all species (p < 0.05) at both 25 and 4 °C, pH 5.6. The relative effect of HNE was greater for myoglobins (Mbs) that contained 12 ± 1 histidine (His) residues than for those that contained 9 His residues (p < 0.05); HNE efficacy in all species except chicken and turkey decreased with time. Mono-HNE adducts were detected in all species except chicken and turkey. In general, HNE alkylation increased the Mbs' ability to accelerate lipid oxidation in a microsome model. However, neither an HNE nor a Mb species dependent effect was observed. Results suggested that microsome model system associated lipid oxidation overshadowed HNE and species effects on OxyMb oxidation observed in lipid-free systems.  相似文献   

9.
Myoglobin (Mb) redox stability affects meat color and is compromised by lipid oxidation products such as 4-hydroxy-2-nonenal (HNE). Pork lipids are generally more unsaturated and would be expected to oxidize readily and produce more oxidation products than beef. Supranutritional supplementation of vitamin E improves Mb redox stability of beef but not pork. The present study investigated HNE-induced redox instability in porcine and bovine myoglobins at 4 degrees C and pH 5.6. Oxymyoglobin (OxyMb) was incubated with HNE (0.075 mM porcine OxyMb + 0.5 mM HNE; 0.15 mM bovine OxyMb + 1.0 mM HNE). In porcine Mb, only monoadducts formed via Michael addition were detected after 72 h, whereas in bovine Mb both mono- and diadducts were identified. LC-MS-MS identified four histidine residues (His 36, 81, 88, and 152) of bovine Mb that were readily adducted by HNE, whereas in porcine Mb only two histidine residues (His 24 and 36) were adducted. These results suggested that the primary structure of bovine Mb predisposes it to greater nucleophilic attack by HNE and subsequent adduction than is suffered by porcine Mb.  相似文献   

10.
Reduction of ferric myoglobin (metmyoglobin, MetMb) to its ferrous form is important for maintaining fresh meat color because only reduced myoglobin can bind oxygen to form the consumer-preferred cherry red color in fresh meat. The objective of this study was to characterize an apparent mitochondria electron transport chain (ETC)-linked pathway for MetMb reduction in vitro. MetMb was reduced in the presence of mitochondria and succinate (p < 0.05); mitochondria or succinate alone did not facilitate MetMb reduction relative to controls (p > 0.05). Flushing samples with oxygen greatly decreased MetMb reduction, while flushing with argon increased MetMb reduction when compared with controls (p < 0.05). ETC inhibitors were used to localize the site where electrons became available for MetMb reduction. MetMb reduction was increased by rotenone addition and decreased by malonic acid (p < 0.05); the reduction was completely abolished by additions of antimycin A or myxothiazol when compared with controls (p < 0.05). These results suggest that electrons become available for MetMb reduction at a site(s) between complex III and IV. Mitochondrial ETC-linked MetMb reduction increased with increased mitochondrial density and succinate concentration (p < 0.05); the greatest MetMb reduction was observed at pH 7.2 and 37 degrees C, and ETC-linked MetMb reducing activity decreased with time postmortem (p < 0.05). These results indicate that ETC-linked MetMb reduction exists but would be minimally active in postmortem muscles.  相似文献   

11.
Natural phenolic antioxidants have been tested in hake (Merluccious merluccious) microsomes as inhibitors of lipid oxidation promoted by fish muscle prooxidants: hemoglobin (Hb), enzymatic NADH-iron and nonenzymatic ascorbate-iron. The phenolics selected were as follows: (a) a grape phenolic extract (OW), (b) a fraction (IV) with isolated grape procyanidins with a medium-low degree of polymerization and galloylation percentage, (c) hydroxytyrosol obtained from olive oil byproducts, and (d) a synthetic phenolic antioxidant, propyl gallate. All compounds delayed lipid oxidation activated by Hb, enzymatic NADH-iron, and nonenzymatic ascorbate-iron, excluding hydroxytyrosol that was not an effective antioxidant on oxidation promoted by nonenzymatic iron. The relative antioxidant efficiency was independent of the prooxidant system, IV > propyl gallate > OW > hydroxytyrosol, and showed a positive correlation with their incorporation into microsomes (p < 0.05). The reducing capacity or ability for donating electrons and the chelating properties may also contribute to the antioxidant activity of phenolics, although these factors were less decisive than their affinity for incorporating into the microsomes. Conversely, the inhibition of Hb oxidation by phenolics and their polarity did not seem to play an important role on antioxidant mechanism. These results stressed the importance of incorporating the exogenous antioxidants into the membranes where are located key substances for fish lipid oxidation (highly unsaturated phospholipids, iron-reducing enzymes, and endogenous alpha-tocopherol).  相似文献   

12.
The hemoglobin variant rHb 0.1, which possesses a decreased ability to form subunits, stimulated lipid oxidation in washed fish muscle less effectively as compared to wild-type hemoglobin (rHb 0.0). This could be due to the lower hemin affinity and more rapid autoxidation rate of subunits as compared to tetramers. To differentiate between hemin affinity and autoxidation effects, ferrous V68T Mb was compared to ferrous wild-type myoglobin (WT Mb). WT Mb has a more rapid hemin loss rate (25-fold) than does V68T, while V68T autoxidized more rapidly than did WT Mb (60-fold). Ferrous WT Mb promoted TBARS and lipid peroxide formation more rapidly than did ferrous V68T (p < 0.01). This indicated hemin loss rate was more critical in determining onset of lipid oxidation as compared to autoxidation rate. Hemin alone was capable of stimulating lipid oxidation. Albumin enhanced the ability of hemin to promote lipid oxidation. MetMb promoted lipid oxidation more effectively than did ferrous Mb, which could be due to the lower hemin affinity of metMb as compared to that of ferrous Mb. EDTA, an iron chelator, had no effect on the rate or extent of lipid oxidation mediated by Mb in the cooked system. Variants with a 975-fold range of hemin affinities promoted lipid oxidation with equivalent efficacy in cooked washed cod contrary to results in uncooked washed cod. The cooking temperatures apparently denature the globin and release hemin reactant to such an extent that the impact of hemin affinity on lipid oxidation observed in the raw state is negated in the cooked state. These studies collectively suggest released hemin is of primary importance in promoting lipid oxidation in raw and cooked washed fish muscle.  相似文献   

13.
This research was aimed at setting up an analytical method for the determination in pork products of 4-hydroxy-2-nonenal (4-HNE), an aldehyde produced from the oxidation of omega-6-polyunsaturated fatty acids. Such a compound mediates various biological effects, but it is considered to be very toxic to mammalian cells at levels higher than physiological ones. The methods used for the determination of 4-HNE in biological fluids, such as blood, were found to be unsuitable for meat samples because both the repeatability and the recovery in spiked samples were unsatisfactory. A new method, based on solid phase extraction and HPLC-MS/MS, was therefore developed and validated. The limit of detection of 4-HNE in spiked samples was 0.043 mg/kg, and the recovery was approximately 60% depending on the concentration. Good linearity was observed in the range of 0.1-10 mg/kg, and repeatability and interday and intraday precision expressed as relative standard deviation were <10%. The method has been successfully applied to the determination of the aldehyde in samples of various pork products. 4-HNE was present in some products, especially the smoked and/or cooked ones, at levels that might not be a real risk for human health.  相似文献   

14.
Peroxynitrite (ONOO(-)), formed from the nearly diffusion limited reaction between nitric oxide and superoxide, could be an important prooxidant in muscle foods. The objective of this study was to determine whether peroxynitrite caused oxidation of pyrogallol red, liposomes, muscle microsomes, and skeletal muscle homogenate. Oxidation of pyrogallol red, liposomes, and microsomes initiated by peroxynitrite continuously produced by 3-morpholinosydnonimine (SIN-1, 2 mM) was time-dependent and enhanced by CO(2) (1 mM). Reagent peroxynitrite (2 mM) caused concentration-dependent oxidation of pyrogallol red, liposomes, and muscle microsomes that was very rapid with no change after 5 min. Peroxynitrite-induced oxidation was suppressed by CO(2) and low pH. Skeletal muscle homogenate oxidized by reagent peroxynitrite (0.5 mM) exhibited gradual oxidation with time and was suppressed by CO(2), low pH, and metal chelators. These data suggest that peroxynitrite could be an important prooxidant in muscle foods.  相似文献   

15.
Effekt of various organic substances on the mobility of fertilizer P Using model trials, studies were carried out on the influence of long-term organic manuring as well as incubation with rotted straw, maize roots, and slurry, on both the diffusion of 32P from added (NH4)2H32PO4 in a ‘soil block’and the fixation rate of fertilizer phosphate in the soil. 1. In the “soil block experiment”, preincubation for 7 days with slurry (added to a low-P brown earth soil at a rate of 5% D. M.) increased diffusion of P as compared to an equivalent amount of added inorganic P. Preincubation with rotted straw resp. maize roots had only insignificant effects. 2. Long-term organic manuring with farmyard manure (P-long-term field trials Weihenstephan) had a significant positive effect on the diffusion of P in the soil block in contrast to annual application of mineral P. Effects of straw manuring were markedly less. 3. Addition of maize roots (5% D. M. to a non P-treated or P-enriched brown earth soil) resulted in a reduced fixation of labelled fertilizer phosphate (by 2–8%), longer preincubation periods, however, increased fixation. Preincubation with rotted straw increased P fixation by 2 to 4% as compared to an equivalent addition of inorganic P (diammonium phosphate).  相似文献   

16.
Ninety, 21-day-old, Japanese quail (Coturnix coturnix japonica) divided into three groups with five subgroups each were fed a basal diet that served as control or a basal diet containing 5 or 10% of dried tomato pulp (DTP), a byproduct of the tomato-processing industry. The DTP contained lycopene and beta-carotene at 281 and 24.3 mg kg(-)(1) of dry weight, respectively. On day 42 of age, birds were slaughtered, and carcasses were trimmed for breast meat. To assess the effect of dietary treatment on the oxidative stability of raw and cooked meat, raw meat was subjected to iron-induced lipid oxidation, whereas both raw and cooked meats were subjected to refrigerated storage at 4 degrees C. The extent of lipid oxidation was determined on the basis of the malondialdehyde (MDA) formed through the use of third-order derivative spectrophotometry. Results showed that after 6 and 9 days of refrigerated storage, MDA values in raw meat were increased. The increase was higher (P < 0.05) for the 10% DTP group and lower (P < 0.05) for the 5% DTP group, compared to control. An analogous oxidation profile was observed for cooked meat at 3, 6, and 9 days of storage. Iron-induced lipid oxidation of raw meat showed that the 10% DTP group as well as the control group exhibited MDA values that did not differ (P > 0.05) from each other at all time points, whereas the 5% DTP group presented MDA values that, although not differing from those of the other groups at 0 and 50 min, were significantly (P < 0.05) lower than those of the other groups at 100 and 150 min of iron-induced lipid oxidation. These results suggested that inclusion of dried tomato pulp in feed at a level of 5% exerted an antioxidant effect, whereas addition at level of 10% exerted a prooxidant effect. Mean alpha-tocopherol levels in the control, 5% DTP, and 10% DTP groups were 2.2, 2.1, and 1.4 mg kg(-)(1) of meat, respectively. Fatty acid analysis showed that the 10% DTP group had a higher (P < 0.05) content of total polyunsaturated fatty acids and a greater (P < 0.05) unsaturated/saturated fatty acid ratio compared to control. There might be an interaction between DTP and alpha-tocopherol that is of importance for the balance between pro- and antioxidative activities. Future experiments should be designed to explore the interaction between individual carotenoids and tocopherols in order to better elucidate their role in oxidative changes.  相似文献   

17.
The metabolism of 3-hydroxy-3-methylindolenine (HMI), a recently discovered metabolite of 3-methylindole (3MI, skatole) produced by porcine liver microsomes, was investigated in vitro using porcine liver cytosol. HMI was rapidly metabolized to a single product, 3-hydroxy-3-methyloxindole (HMOI), by porcine cytosol. By the use of the selective inhibitors menadione and quinacrine, it was shown that the enzyme responsible for the oxidation of HMI into HMOI was aldehyde oxidase (AO; aldehyde:oxygen oxidoreductase, EC 1.2.3.1). The activity of AO in the conversion of HMI to HMOI was measured in a population of pigs (n = 30) with a wide range of 3MI levels in back fat (0.07-0.30 mg/kg). AO activity was found to be negatively correlated (r = -0.70; P < 0.001) with the level of 3MI in fat. The results of the present study suggest that AO plays an important role in the metabolism of 3MI in the pig and that its catalytic activity is related to an adequate 3MI clearance.  相似文献   

18.
The oxidation of alpha-tocopherol (TH) in beef was analyzed using a stable isotope dilution capillary gas chromatography-mass spectrometry assay. TH decreased while alpha-tocopherolquinone (TQ) and 2,3-epoxy-alpha-tocopherolquinone (TQE(2)) increased in ground longissimus lumborum (LL) and psoas major (PM) muscles during storage (P < 0.10). In LL steaks, the relative concentrations of TH decreased and TQ and TQE(2) increased in surface samples; changes were less dramatic in deep samples. Deuterated alpha-tocopherolhydroquinone (THQ) standard was not recovered and endogenous THQ was not detected in meat; THQ was measurable in microsomes isolated from PM and incubated in the presence of 2, 2'-azobis(2-amidopropane)HCl (ABAP) or myoglobin. ABAP-challenged microsomes yielded a tocopherol product profile which favored 5, 6-epoxy-alpha-tocopherolquinone (TQE(1)) and TQE(2), while the use of myoglobin as prooxidant resulted in a higher proportion of TQ and THQ. Results demonstrated that concentrations of TH decreased and TQ and TQE(2) increased in meat during storage and are consistent with the peroxy-radical scavenging function of tocopherol.  相似文献   

19.
基于高光谱技术的猪肉肌红蛋白含量无损检测   总被引：2，自引：2，他引：0  
为充分利用猪肉光谱与图像信息,实现猪肉肌红蛋白含量的在线检测,该研究提出一种基于深度学习模型的猪肉肌红蛋白含量无损检测方法。采用高光谱设备采集冷藏过程中猪肉高光谱图像,通过ENVI5.3选择图像感兴趣区域（Region Of Interest,ROI）,分别提取ROI平均光谱信息与主成分图像信息。利用卷积自动编码器（Convolutional Auto Encoder,CAE）提取光谱与图像信息深度特征,分别建立光谱特征、图像特征及图-谱融合特征与肌红蛋白含量之间关系的卷积神经网络（Convolutional Neural Network,CNN）预测模型。其中基于融合深度特征CNN预测模型准确度较高,该模型对脱氧肌红蛋白（DeoMb）、氧合肌红蛋白（OxyMb）、高铁肌红蛋白（MetMb）含量预测集决定系数分别为：0.964 5、0.973 2、0.958 5,预测集均方根误差 RMSEP分别为：0.015 8、0.226 6、0.381 6。为进一步验证图-谱融合特征与猪肉肌红蛋白存在对应关系,分别建立偏最小二乘回归（Partial Least Squares Regression,PLSR）、支持向量机回归（Support Vector Regression,SVR）预测模型。结果表明：CAE能充分提取图像与光谱特征;基于融合特征建立回归模型能提高肌红蛋白含量预测精度,相比于光谱信息与图像信息,以MetMb为例其分别提高5.42%、16.12%。该检测方法为肉类质量在线检测提供参考,具有好的应用前景。  相似文献   

20.
The xylose-fermenting yeast Candida intermedia produces two isoforms of xylose reductase: one is NADPH-dependent (monospecific xylose reductase; msXR), and another is shown here to prefer NADH approximately 4-fold over NADPH (dual specific xylose reductase; dsXR). To compare the functional properties of the isozymes, a steady-state kinetic analysis for the reaction d-xylose + NAD(P)H + H(+) <--> xylitol + NAD(P)(+) was carried out and specificity constants (k(cat)/K(aldehyde)) were measured for the reduction of a series of aldehydes differing in side-chain size as well as hydrogen-bonding capabilities with the substrate binding pocket of the enzyme. dsXR binds NAD(P)(+) (K(iNAD+) = 70 microM; K(iNADP+) = 55 microM) weakly and NADH (K(i) = 8 microM) about as tightly as NADPH (K(i) = 14 microM). msXR shows uniform binding of NADPH and NADP(+) (K(iNADP+) approximately K(iNADPH) = 20 microM). A quantitative structure-activity relationship analysis was carried out by correlating logarithmic k(cat)/K(aldehyde) values for dsXR with corresponding logarithmic k(cat)/K(aldehyde) values for msXR. This correlation is linear with a slope of approximately 1 (r (2) = 0.912), indicating that no isozyme-related pattern of substrate specificity prevails and aldehyde-binding modes are identical in both XR forms. Binary complexes of dsXR-NADH and msXR-NADPH show the same macroscopic pK of approximately 9.0-9.5, above which the activity is lost in both enzymes. A lower pK of 7.4 is seen for dsXR-NADPH. Specificity for NADH and greater binding affinity for NAD(P)H than NAD(P)(+) are thus the main features of enzymic function that distinguish dsXR from msXR.  相似文献   

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