Normalisation of gait EMGs: a re-examination

The purpose of this study was to compare four different methods of normalising electromyograms (EMGs) recorded during normal gait. Comparisons were made between the amplitude, intra-individual variability and inter-individual variability of EMGs. Surface EMGs were recorded from the biceps femoris, semitendinosus, vastus lateralis and vastus medialis of ten males and two females while they walked on a treadmill at a self-selected speed. EMGs from the same muscles were subsequently recorded during isometric maximal voluntary contractions (MVCs) and concentric, isokinetic MVCs that were performed between 0.52 and 7.85 rad·s⁻¹ on a BIODEX dynamometer. EMGs were also recorded during eccentric, isokinetic MVCs between 0.52 and 2.62 rad·s⁻¹. Gait EMGs were then normalised at 2% intervals of the gait cycle by expressing them as a percentage of the following reference values: the mean (mean dynamic method) and the peak (peak dynamic method) EMG from the intra-individual ensemble average; the EMG from an isometric MVC (isometric MVC method); and the EMG from an isokinetic MVC that occurred with the same muscle action, length and velocity of musculotendinous unit as the gait EMGs (isokinetic MVC method). The isokinetic MVC method produced significantly greater (P<0.05) intra-individual variability compared to the other methods when it was measured using the variance ratio. Inter-individual variability of gait EMGs, again measured using the variance ratio, was also greatest when they were normalised using the isokinetic MVC method. The pattern and amplitude of EMGs normalised using the isometric MVC method and the isokinetic MVC method were very similar (root mean square difference and absolute difference both less than 3%). It was concluded that the isokinetic MVC method should not be adopted by gait researchers or clinicians as it does not reduce intra- or inter-individual variability anymore than existing normalisation methods, nor does it provide a more representative measure of muscle activation during gait than the isometric MVC method.     

Agonist and antagonist muscle activation during maximal and submaximal isokinetic fatigue tests of the knee extensors

The purpose of this study was to examine the differences in electromyographic activity of agonist and antagonist knee musculature between a maximal and a submaximal isokinetic fatigue protocol. Fourteen healthy males (age: 24.3 ± 2.5 years) performed 25 maximal (MIFP) and 60 submaximal (SIFP) isokinetic concentric efforts of the knee extensors at 60° s⁻¹, across a 90° range of motion. The two protocols were performed a week apart. The EMG activity of vastus medialis (VM), vastus lateralis (VL) and biceps femoris (BF) were recorded using surface electrodes. The peak torque (PT) and average EMG (aEMG) were expressed as percentages of pre-fatigue maximal value. One-way analysis of variance indicated a significant (p < 0.05) decline of PT during the maximal (45.7%) and submaximal (46.8%) protocols. During the maximal test, the VM and VL aEMG initially increased and then decreased. In contrast, VM and VL aEMG continuously increased during submaximal testing (p < 0.05). The antagonist (BF) aEMG remained constant during maximal test but it increased significantly and then declined during the submaximal testing. The above results indicate that agonist and antagonist activity depends on the intensity of the selected isokinetic fatigue test.     

Differential control during maximal concentric and eccentric loading revealed by characteristics of the electromyogram

Maximal eccentric loading has been associated with higher levels of spindle afferent activity but lower levels of integrated EMG as compared to maximal concentric loading. Elbow flexor EMG was recorded from 17 subjects during concentric (CONC) and eccentric (ECC) elbow flexion at 70° s⁻¹ using a Kin-Com dynamometer. We hypothesized that peak EMG amplitude would be more sensitive to fluctuations in facilitation by the spindle primary afferents via the segmental stretch reflex pathway, and that the mean EMG would be more reflective of the ongoing level of muscle activation. A ratio of peak to mean EMG (P/M EMG ratio) was predicted to be larger during maximal eccentric loading than maximal concentric loading. The peak EMG (P<0.013) and the P/M EMG ratio (P<0.001) were significantly greater during the ECC condition than the CONC condition. In a subgroup of three subjects who underwent 3 weeks of eccentrically biased weight training, EMG, peak torque and torque variability were assessed before and after training. P/M EMG ratio decreased, while peak torque and torque variability increased following the training. Differences in the P/M EMG ratio appear to reflect differences in the way eccentric and concentric muscle actions are controlled and do not simply represent less control during the eccentric task.     

Factors predicting knee function in patients with meniscal lesions

The relationships between the magnitude of strength and muscle activation (EMG activity) deficits during maximal isokinetic knee movements and clinical and anthropometric factors (pain, effusion, type of meniscal lesion, degenerative changes, duration of symptoms and age) were studied in a group of 34 patients 5 days (5.3 ± 6.2 days) before arthroscopic meniscectomy. Moderate correlations were found between deficits in the area under the torque-angle curve (work) and the activation level in the agonist muscles during both the extension and flexion tests at 30° s⁻¹ and 180° s⁻¹ suggesting that the decrease in the isokinetic mechanical output is dependent on the reduced muscle activation. The severity of pain during tests was the best predictive clinical factor for the size of the work and EMG deficits but each of the tollowing factors: the presence of knee effusion and thigh atrophy, the type of meniscal lesion and the age of the patients, also had some predictive value for the magnitude of the deficits. In contrast, the duration of symptoms and the presence of osteo-articular degenerative changes were not related to the preoperative isokinetic knee performance. These results suggest that older patients (especially over 45 yr) with bucket-handle meniscal tears, knee effusion, thigh atrophy and severe pain on exertion, are most likely to have poor preoperative knee isokinetic performance. Since the magnitude of the pre- and postoperative knee deficits are correlated, it is important to identify patients at risk preoperatively so that optimal rehabilitation can be instituted.     

Rate of electron transfer between plastocyanin, cytochrome ƒ, related proteins and artificial redox reagents in solution

The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10⁷ M⁻¹ · s⁻¹, at 298 °K and pH 7.0, with activation parameters ΔH^‡ = 44 kJ · mole⁻¹ and ΔS^‡ = +46 J · mole⁻¹ · °K⁻¹. Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10⁵, 7.5 · 10⁵ and 1.0 · 10⁶ M⁻¹ · s⁻¹, respectively.

Similar measurements made with azurin instead of plastocyanin gave k = 6 · 10⁶ and approx. 2 · 10⁷ M⁻¹ · s⁻¹ for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively.

Rate constants of 115 and 80 M⁻¹ · s⁻¹ were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 10⁴ and 8 · 10⁴ M⁻¹ · s⁻¹.

The results are discussed in relation to photosynthetic electron transport.     

Contraction mode shift in quadriceps femoris muscle activation during dynamic knee extensor exercise with increasing loads

The objective of the present study was to examine the superficial quadriceps femoris (QF) muscle electromyogram (EMG) during dynamic sub-maximal knee extension exercise between young adult men and women. Thirty subjects completed, in a random order, 2 sub-maximal repetitions of single-leg knee extensions at 20-90% of their one-repetition maximum (1RM). Vastus medialis (VM), vastus lateralis (VL) and rectus femoris (RF) muscle integrated EMG (IEMG) during each sub-maximal lift was normalized to the respective 1RM for concentric, isometric and eccentric modes. The EMG median frequency (f(med)) was determined over the isometric mode. Men attained a significantly (p<0.05) greater knee angular velocity than the women during the concentric mode (83.6+/-19.1 degrees /s and 67.4+/-19.8 degrees /s, respectively). RF IEMG was significantly lesser than the VM (p=0.014) and VL (p<0.001) muscles, when collapsed across all contraction modes, loads, and sex. Overall IEMG was significantly greater during the concentric (p<0.001) and isometric (p<0.001) modes, than the eccentric mode. Men generated significantly (p=0.03) greater VL muscle IEMG than the women, while the opposite pattern emerged for the RF muscle. VM f(med) (105.1+/-11.1Hz) was significantly lesser than the VL (180.3+/-19.5Hz) and RF (127.7+/-13.9Hz) muscles across all lifting intensities, while the men (137.7+/-10.7Hz) generated greater values than the women (129.0+/-11.4Hz). The findings demonstrate a reduction in QF muscle activation across the concentric to eccentric transition, which may be related to the mode-specific velocity pattern.     

Different neuromuscular recruitment patterns during eccentric, concentric and isometric contractions

Aim. The purpose of this study was to determine the neuromuscular fatigue profiles during 100 s isometric (ISO), concentric (CON), and eccentric (ECC) activity.

Methods. Twelve subjects (age 25.1±3.7 years, mass 70.1±8.2 kg, mean±SD) performed ISO, CON and ECC maximal voluntary contractions and 100 s endurance trials on an isokinetic dynamometer. Raw EMG data were recorded throughout each trial from the rectus femoris of the right limb. Corresponding data for integrated electromyography (IEMG), percentile frequency shifts (MPFS) and peak torque output were divided into five 5 s epochs and subsequently normalised with the first epoch being the reference point, in order to assess changes over time.

Results. There were no significant differences between ECC, CON and ISO peak torque output (211±63 vs 169±41 vs 177±61 Nm; ECC, CON, ISO) and IEMG activity (280±143 vs 305±146 vs 287±143 mV; ECC, CON, ISO) during maximal contractions. Serial reductions in torque output were greatest in ISO in which torque output during the final epoch was 31±13% of initial values, similar to the final torque values in CON (58±15%), but significantly less than ECC (108.6±38.6%; P<0.001) values. In CON and ECC, IEMG was maintained (95±27% and 93±21%; CON and ECC), whereas IEMG for ISO decreased to 38±13% of initial values. The greatest reduction in MPFS occurred in CON (69±10%) compared to ISO (78±9%; P<0.05) and ECC (93±6%; P<0.001).

Conclusion. These data demonstrate distinct neuromuscular fatigue profiles for the different types of muscle contraction. Whereas eccentric activity was largely fatigue resistant, isometric and concentric contractions displayed different neuromuscular fatigue profiles.     

The effects of interelectrode distance over the innervation zone and normalization on the electromyographic amplitude and mean power frequency versus concentric,eccentric, and isometric torque relationships for the vastus lateralis muscle

The purpose of this study was to examine the influence of interelectrode distance (IED) over the estimated innervation zone (IZ) for the vastus lateralis muscle and normalization on the torque-related patterns of responses for electromyographic (EMG) amplitude and mean power frequency (MPF) during concentric isokinetic, eccentric isokinetic, and isometric muscle actions of the leg extensors. Eight men performed submaximal to maximal concentric isokinetic, eccentric isokinetic, and isometric muscle actions of the dominant leg extensors. Surface EMG signals were recorded simultaneously with two bipolar electrode arrangements in single differential configuration (20 and 40 mm IEDs) placed over the estimated IZ for the vastus lateralis muscle and a third electrode arrangement in single differential configuration (20 mm IED) placed distal to the estimated IZ. The results indicated that there were only a few (six of 90 statistical comparisons) significant (p < 0.05) mean differences among the three electrode arrangements for absolute EMG amplitude. There were no mean differences among the three electrode arrangements for absolute or normalized EMG MPF values or normalized EMG amplitude for the three types of muscle actions. Thus, it may be possible to reduce the potential influence of the IZ on amplitude and spectral parameters of the EMG signal through normalization.     

Co-activation of vastus lateralis and biceps femoris muscles in pubertal children and adults

Determining the mechanisms of co-activation around the knee joint with respect to age and sex is important in terms of our greater understanding of strength development. The purpose of this study was to examine the effects of age, sex and muscle action on moment of force and electromyographic (EMG) activity of the agonist and antagonist muscle groups during isokinetic eccentric and concentric knee extension and flexion. The study comprised nine pubertal boys [mean age 12.6 (SD 0.5) years], nine girls [12.7 (SD 0.5) years] nine adult men [23.1 (SD 2.1) years] and nine adult women [23.7 (SD 3.1) years] who performed maximal isometric eccentric and concentric efforts of knee extensors and flexors on a dynamometer at 30 degrees x s(-1). The moment of force and surface EMG activity of vastus lateralis and biceps femoris muscles were recorded. The moment of force:agonist averaged EMG (aEMG) ratios were calculated. The antagonist aEMG values were expressed as a percentage of the aEMG activity of the same muscle, at the same angle, angular velocity and muscle action when the muscle was acting as agonist. Three-way analysis of variance (ANOVA) designs indicated no significant effects of age or sex on moment:aEMG ratios. Eccentric ratios were significantly higher than the corresponding concentric ones (P < 0.05). The results also indicated no significant effect of age and sex on the aEMG of the vastus lateralis and biceps femoris muscles when acting as antagonists. The antagonist aEMG was significantly greater during concentric agonist efforts compared with the corresponding eccentric ones (P < 0.05). These findings would suggest that the moment exerted per unit of agonist EMG and the antagonist activity are similar in children compared with adults and are not sex dependent. Future comparisons between eccentric and concentric moments of force and agonist ENG should take into consideration the antagonist effects, irrespective of age or sex.     

The effects of antagonist moment on the resultant knee joint moment during isokinetic testing of the knee extensors

The purpose of this study was to examine the effects of moment of antagonistic muscle on the resultant joint moment during isokinetic eccentric and concentric efforts of the knee extensors. Ten males performed maximum eccentric and concentric knee extension and flexion efforts on a Biodex dynamometer at 0.52 rad · s⁻¹ (30° · s⁻¹). Electromyographic (EMG) activity of vastus medialis and biceps femoris (hamstrings) was also recorded. The antagonistic moment of the hamstrings was determined by recording the integrated EMG (iEMG)/moment relationship at different levels of muscle effort. The iEMG/moment curves were fitted using second-degree polynomials. The polynomials were then used to predict the antagonistic moment exerted by the hamstrings from the antagonist iEMG. The antagonistic moment had a maximum of 42.92 Nm and 28.97 Nm under concentric and eccentric conditions respectively; paired t-tests indicated that this was a significant difference (P < 0.05). These results indicate that the resultant joint moment of knee extensors is the result of both agonist and antagonist muscle activation. The greater antagonist muscle activity under concentric activation conditions may be partly responsible for the lower resultant joint concentric moment of knee extensors compared with the corresponding eccentric activation. The antagonist moment significantly affects comparisons between the isokinetic moments and agonist EMG and in vitro force measurements under different testing (muscle action and angular velocity) conditions. Accepted: 25 February 1997     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

.     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

Cytochrome oxidase from Pseudomonas aeruginosa. IV. Reaction with oxygen and carbon monoxide

The reaction between a cytochrome oxidase from Pseudomonas aeruginosa and oxygen has been studied by a rapid mixing technique. The data indicate that the heme d₁ moiety of the ascorbate-reduced enzyme is oxidized faster than the heme c component. The oxidation of heme d₁ is accurately second order with respect to oxygen and has a rate constant of 5.7 · 10⁴ M⁻¹ · s⁻¹ at 20 °C. The oxidation of the heme c has a first-order rate constant of about 8 s⁻¹ at infinite concentration of O₂. The results indicate that the rate-limiting step is the internal transfer of electrons from heme c to heme d₁. These more rapid reactions are followed by more complicated but smaller absorbance changes whose origin is still not clear.

The reaction of ascorbate-reduced oxidase with CO has also been studied and is second order with a rate constant of 1.8 · 10⁴ M⁻¹ · s⁻¹. The initial reaction with CO is followed by a slower reaction of significantly less magnitude. The equilibrium constant for the reaction with CO, calculated as a dissociation constant from titrimetric experiments with dithionite-reduced oxidase, is about 2.3 · 10⁻⁶ M. From these data a rate constant of 0.041 s⁻¹ can be calculated for the dissociation of CO from the enzyme.     

Isokinetic elbow flexion and coactivation following eccentric training.

The influence of an eccentric training on torque/angular velocity relationships and coactivation level during maximal voluntary isokinetic elbow flexion was examined. Seventeen subjects divided into two groups (Eccentric Group EG, n = 9 Control Group CG, n = 8) performed on an isokinetic dynamometer, before and after training, maximal isokinetic elbow flexions at eight angular velocities (from - 120 degrees s(-1) under eccentric conditions to 240 degrees s(-1) under concentric conditions), and held maximal and submaximal isometric actions. Under all conditions, the myoelectric activities (EMG) of the biceps and the triceps brachii muscles were recorded and quantified as the RMS value. Eccentric training of the EG consisted of 5x6 eccentric muscle actions at 100 and 120% of one maximal repetition (IRM) for 21 sessions and lasted 7 weeks. In the EG after training, torque was significantly increased at all angular velocities tested (ranging from 11.4% at 30 degrees (s-1) to 45.5% at - 120 degrees s(-1)) (p < 0.05). These changes were accompanied by an increase in the RMS activities of the BB muscle under eccentric conditions (from - 120 to - 30 degrees (s-1)) and at the highest concentric angular velocities (180 and 24 degrees s(-1)) (p < 0.05). The RMS activity of the TB muscle was not affected by the angular velocity in either group for all action modes. The influence of eccentric training on the torque gains under eccentric conditions and for the highest velocities was attributed essentially to neural adaptations.     

Viscoelastic properties of passive skeletal muscle in compression: stress-relaxation behaviour and constitutive modelling

The compressive properties of skeletal muscle are important in impact biomechanics, rehabilitation engineering and surgical simulation. However, the mechanical behaviour of muscle tissue in compression remains poorly characterised. In this paper, the time-dependent properties of passive skeletal muscle were investigated using a combined experimental and theoretical approach. Uniaxial ramp and hold compression tests were performed in vitro on fresh porcine skeletal muscle at various rates and orientations of the tissue fibres. Results show that above a very small compression rate, the viscoelastic component plays a significant role in muscle mechanical properties; it represents approximately 50% of the total stress reached at a compression rate of 0.5% s⁻¹. A stiffening effect with compression rate is observed especially in directions closer to the muscle fibres. Skeletal muscle viscoelastic behaviour is thus dependent on compression rate and fibre orientation.

A model is proposed to represent the observed experimental behaviour, which is based on the quasi-linear viscoelasticity framework. A previously developed strain-dependent Young's Moduli formulation was extended with Prony series to account for the tissue viscoelastic properties. Parameters of the model were obtained by fitting to stress-relaxation data obtained in the muscle fibre, cross-fibre and 45° directions. The model then successfully predicted stress-relaxation behaviour at 60° from the fibre direction (errors <25%). Simultaneous fitting to data obtained at compression rates of 0.5% s⁻¹, 1% s⁻¹ and 10% s⁻¹ was performed and the model provided a good fit to the data as well as good predictions of muscle behaviour at rates of 0.05% s⁻¹ and 5% s⁻¹ (errors <25%).     

Reduction of ferricytochrome c by hydrogen atoms: Evidence for intramolecular transfer of reducing equivalent

Direct evidence obtained by means of the technique of pulse radiolysis-kinetic spectrometry, with measurements in the time range 10⁻⁶ to 1 s, is presented that, consequent upon reaction of a single H-atom with a single molecule of ferricytochrome c, a reducing equivalent is transmitted via the protein structure to the ferriheme moiety. Such transmission accounts for at least 70% of the total reduction of the ferri to the ferro state of cytochrome c. The remainder of the total reduction takes place without stages resolvable on the time scale of these experiments. Reduction brought about by H atoms appears to follow a different course than reduction by hydrated electrons. In the latter case, intramolecular transmission of reducing equivalents could not be demonstrated (Lichtin, N. N., Shafferman, A. and Stein, G. (1973) Biochim. Biophys. Acta 314, 117–135).

Not every H-atom reacts with ferricytochrome c at a site which results in conversion of the Fe(III) state to the Fe(II) state. Approximately half of reacting H-atoms do not produce reduction.

The following second order rate constants have been determined in solutions of low ionic strength at 20±2 °C: k[H+ferricytochrome c] = (1.0±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0 and 6.7; k[H+ferrocytochrome c] = (1.3±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0; k[e⁻_aq + ferrocytochrome c] = (1.9±0.4) · 10¹⁰ M⁻¹ · s⁻¹ at pH 6.7.     

Pulse radiolysis kinetics of the reaction of the hydrated electron and the carboxyl anion radical with Pseudomonas aeruginosa cytochrome c-551

The kinetics of the reaction of hydrated electron (e⁻_aq) and carboxyl anion radical (CO₂) with Pseudomonas aeruginosa ferricytochrome c-551 were studied by pulse radiolysis. The rate of reaction of e⁻_aq with the negatively charged ferricytochrome c-551 (17 nM⁻¹ · s⁻¹) is significantly slower than the larger positively charged horse heart ferricytochrome c (70 nM⁻ · s⁻). This difference cannot be explained solely by electrostatic effects on the diffusion-controlled reactions. After the initial encounter of e⁻_aq with the protein, ferricytochrome c-551 is less effective in transferring an electron to the heme which may be due to the negative charge on the protein. The charge on ferricytochrome c-551 is estimated to be −5 at pH 7 from the effect of ionic strength on the reaction rate. A slower relaxation (2 · 10⁴ s⁻¹) observed after fast e⁻_aq reduction is attributed to a small conformational change. The rate of reaction of CO₂ with ferricytochrome c-551 (0.7 nM⁻¹ · s⁻) is, after electrostatic correction, the same as ferricytochrome c, indicating that the steric requirements for reaction are similar. This reaction probably takes place through the exposed heme edge.     

Biochemical and biophysical studies on cytochrome c oxidase XIV. The reaction with cytochrome c as studied by pulse radiolysis

1. The reduction of cytochrome c oxidase by hydrated electrons was studied in the absence and presence of cytochrome c.

2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent.

3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons (k = 4 · 10¹⁰ M⁻¹ · s⁻¹ at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 10⁷ M⁻¹ · s⁻¹ at 22 °C and pH 7.2.

4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase.

5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28.     

Butylmalonate is a transition state analogue for aminocylase I

Butylmalonate (butyl propanedioic acid) is a slow-binding inhibitor of porcine renal aminoacylase I (EC 3.5.1.14), causing transients of activity with half-times of more than 10 min. At 25°C and pH 7.0, the dissociation rate of the complex is approximately 6 × 10⁻⁴ s⁻¹, while the rate constant of complex formation is in the order of 20 M⁻¹·s⁻¹. In good agreement with these data, steady-state kinetics yield an estimated inhibition constant around 100 μM. Molecular mechanics calculations showed that conformation and charge distribution of butylmalonate are strikingly similar to those of the putative transition state of aminoacylase catalysis.