Mercaptoethanol rates

The aqueous ferricyanide oxidation of 2-mercaptoethanol to the disulphide is also complex kinetically" . In the pH range used (l.S. l) no complication from ionisation of the thiol is expected. Individual decays of oxidant concentrations are initially second-order but eventually become almost zero-order. For both second-and zero-order paths the rate depends on the first power of the thiol concentration and the former path is retarded by increasing the acidity, an approximately inverse relation existing above pH 3.2. Addition of ferrocyanide transforms the kinetics the rapid, second-order path is inhibited and the zero-order path is accelerated until, at 10 M ferrocyanide, the whole of the disappearance of oxidant is zero-order. Addition of Pb(C104)2, which removes product ferrocyanide, greatly enhances the oxidation rate and the consumption of oxidant becomes rs/-order. Two routes are considered to co-exist (taking due account of the acidity of ferrocyanic acid), viz. 

Wash particles (e.g., 100 mg of 1 pm carboxylated latex beads) into coupling buffer (i.e., 50 mM MES, pH 6.0 or 50 mM sodium phosphate, pH 7.2 buffers with pH values from pH 4.5 -7.5 may be used with success however, as the pH increases the reaction rate will decrease). Suspend the particles in 5 ml coupling buffer. The addition of a dilute detergent solution may be done to increase particle stability (e.g., final concentration of 0.01 percent sodium dodecyl sulfate (SDS)). Avoid the addition of any components containing carboxylates or amines (such as acetate, glycine, Tris, imidazole, etc.). Also, avoid the presence of thiols (e.g., dithiothreitol (DTT), 2-mercaptoethanol, etc.), as these will react with EDC and effectively inactivate it. 

Second-order rate constants for MTSEA-, MTSET-, and MTSES-modification of residues within the loop D region of the GABA-binding site. Second-order rate constants (IC2) represent the mean standard deviation. NR, no reaction. The free solution rates were reported by Karlin and Akabas (1998) and reflect the rates of MTS reaction with 2-mercaptoethanol. Adapted from Holden and Czajkowski (2002) with permission from the American Society of Biochemistry and Molecular Biology... 

Figure 2. Illustration of the importance of the choice of reaction conditions on the determination of initial velocity. Shown are four conditions applied to examine the rate behavior of Escherichia coli NAD+-dependent coenzyme A-linked aldehyde dehydrogenase (Reaction NAD+ + CoA-SH + Acetaldehyde = NADH + Acetyl-S-CoA + H+). All assay mixtures contained enzyme, 0.5 mM NAD+, 8 /jlW CoA-SFI, 16 mM acetaldehyde, and 22.5 mM Tris buffer at pFI 8.1. (a) Time-course observed when enzyme was added to the standard assay (b) time-course observed when enzyme was added to standard assay augmented with 10 mM 2-mercaptoethanol (c) time-course observed when enzyme was first preincubated for 15 min with 8 /jlW CoA-SH, 16 mM acetaldehyde, 10 mM 2-mercaptoethanol, and 22.5 mM Tris buffer at pH 8.1, and the reaction was initiated by addition of NAD+ (d) time-course observed when enzyme was preincubated with lOmM 2-mercaptoethanol for 15 min andthen addedtostandard assay augmented with 10 mM 2-mercaptoethanol. The data are most compatible with the idea that the enzyme has an active-site thiol group that must be reduced to express full catalytic activity during assay.

Fig. 12. Diagram of elution pattern of red cell acid phosphatase and various markers on Biogel P 60. The position of the various protein markers was determined both by optical density determination and by starch gel electrophoresis of the individual fractions (83). The experiment was carried out using a polyacrylamide gel (Biogel P 60, 50-150 mesh exclusion limit >60,000 Bio-Rad Laboratories, California) in 0.05 M tris buffer, pH 8.0, containing 0.08% (v/v) Tween 80 and 0.1% (v/v) 2-mercaptoethanol to stabilize the enzyme. Column 60 X 4 cm. Flow rate 20 ml/hr, 4 ml fractions. (A) OD at 280 nm, ( ) OD at 540 nm, ( ) LDH assay with p-nitrophenyl phosphate for AcP. From Hopkinson and Harris (85).

Equation 7 applies for thiolate anion addition. Ethylene oxide is nearly three times more sensitive than 1 to the nucleophilicity of the thiol. The second-order rate constants for the reactions of 2-mercaptoethanol with 1,47, and 48 are 3.32, 1.58, and 2.04M"1 sec-1, respectively. This shows that 1 is only slightly more reactive and that the K-region does not necessarily make the... 

Triggered by a thiol, they undergo very amusing rearrangements leading to biradicals. Examples are shown in reactions (58) and (59) and reactions (61) and (62). For NCS and 2-mercaptoethanol, the trigger rate constant is 4 dm3 mol-1 s 1 at pH 7.8 (Povirk and Goldberg 1983). 

Tsanaclis et al. [33] described a reversed-phase HPLC method for determination of vigabatrin in plasma after derivatization with phthalal-dehyde. Serum was mixed with y-amino-y-phenylbutyric acid and methanol. Derivatization was carried out with phthaldehyde in borate buffer (pH 9.5) containing 2-mercaptoethanol. The resulting mixture was separated on a column (15 cm x 4.6 mm) of C18 Microsorb (5 /an) with 10 M H3P04-acetonitrile-methanol (6 3 1) as mobile phase with a flow rate of 2 ml/min and fluorimetric detection between 418 and 700 nm (excitation at 370 nm). The detection limit was 0.08 fig/ml of vigabatrin. The CVs were 9%, 5%, and 5% at 2.14, 20.1, and 83.61 fig/ml/min, respectively. Common anticonvulsant did not interfere. 

Ratnaraj and Patsalos [37] described an HPLC method for simultaneous determination of vigabatrin and gabapentin in human serum after precolumn derivatization with o-phthaldialdehyde in the presence of mercaptoethanol and fluorimetric detection at 440 nm with excitation at 340 nm. Separation was achieved on a Hypersil BDS C18 (3 /tm) column (12.5 cm x 3 mm) with a mixture of 250 mM phosphate buffer, acetonitrile, water, and methanol as a gradient mobile phase (flow rate 0.45 ml/min). The method was linear over the concentration range of 25-400 yg/ml for vigabatrin. The lower limit of detection was 1 /(M for both analytes. Within- and between-batch RSD were 2-i% and 3-4%, respectively. No interference from other commonly prescribed antiepileptic drugs was observed. 

While azides were far more susceptible to reduction by dithiols, the rates of reduction of a diazotrifluoropropionyl derivative by dithiothreitol, (3-mercaptoethanol, cysteine, and reduced glutathione did not differ widely. Thioglycolic acid was however a poor reductant and it was suggested that it should be used to replace (3-mercaptoethanol or DTT when diazo reagents are used. The reduction may be monitored by TLC or by a 500-fold increase in the absorbance at 260 nm. 

Many /x-oxo dimers are not stable under reducing conditions owing to instability of the n-oxo bridge moiety in either the mixed-valence or diferrous states. For example, [Fe(HBpz3)]20(0Ac)2 (HBpz3 is hydro-tris(pyrazolyl)borate) shows an electrochemical irreversible reduction wave even at very high scan rates (5 V/s) (13). The fact that the ascorbic acid does not show the same reactivity as 2-mercaptoethanol may be a consequence of differences in the redox potentials or the ability of the reductant to displace ligands and coordinate to the metal center. 

Figure 4. Inactivation of trypsin (0) and trypsin-dextran conjugate (O) at 37°C and pH 8.1 in 8M urea and 5mM 2-mercaptoethanol. The broken line shows the rate of inactivation of trypsin-dextran conjugate after dextranase treatment.

Especially useful is the reaction of carbodiimides with 2-mercaptoethanol because rapid reaction with EDC occurs with rates similar to the reaction of carboxyl groups with EDC. °... 

The rate of acrylamide polymerization initiated by the K2S04/2-mercaptoethanol redox system is of first order with respect to monomer concentration and (3/2)-th order with respect to K2SO+ concentration, the overall energy of activation being 134kJ mol-1. 

The rate constants of repair reactions of pyrimidinyl radicals of various structures (Pyr ) by different thiols — RSH = cysteamine (CysAmSH), 2-mercaptoethanol (MerSH), cysteine (CysSH) and penicillamine (PenSH) — were determined by means of pulse radiolysis in aqueous and alcohol-containing solutions. 

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