PH-stat titrations

Marsh grapefhiit (MGF) pulp was homogenized in 5 volumes of extraction buffer at 4 C and maintained at pH 8.0 (28). The homogenate was stirred for one hour, centrifuged and the supernatant used as the PE extract. Activity was measured by titration with a Brinkman (Westbury, NY) pH stat titrator at pH 7.5 and 30°C in 25 mL of 1 % high methoxyl pectin (Citrus Colloids Limited, Hereford, UK) with O.IM NaCl. PE units are expressed as the microequivalents of ester hydrolysed per minute. Uronic acid analyses were conducted based on the m-phenyl phenol (4) as modified for microplate reading (30). 

The titrant used in pH-stat titration is usually a dilute (perhaps 0.01 M) acid or base. Two different sets of data can be obtained from a pH-stat titration. The amount of a substrate consumed or product formed can be determined by the total amount of titratant used. Because the titrant is added over a period of time, the rate of reaction can also be determined. 

Ficara E, Rozzi A, Cortelezzi P. Theory of pH-stat titration. Biotechnol. Bioeng. 2003 82 28-37. 

Protein Modification. Acylation was performed at room temperature by adding dropwise the anhydride to the protein solution (2 mg/ml). To reach the maximum level of modification, three equivalents of anhydride per lysine were needed. The pH was kept at 9 during the reaction by a controlled and continuous addition of 0.5 M NaOH using an automated pH-stat titration device. The reaction was considered to be complete when the pH of the reaction medium remained constant. To remove salts and excess reagents, the protein solution was dialyzed exhaustively against water and then lyophilized. 

This investigation was mainly concerned with the influence of water content on the rate of dealkalization at 50 C and a pH of 8. Further experimentation centered around dealkalization variables such as temperature, pH, leach solution concentration and time. This was accomplished by using a Metrohm End Point/pH Stat Titrator. The predetermined pH was controlled by automatic addition of titrant to the leach solution. 

A titrametric assay of PLCSc, alternatively called the pH-stat method, was the workhorse in early studies [28]. This method simply involves titrating the acidic product of the PLC reaction as it is formed with a solution of standard base. An advantage of this continuous assay is that it can be used to detect the turnover of both synthetic and natural substrates, and its sensitivity has been estimated to be in the 20-100 nmol range. However, the pH-stat assay has low throughput capability, and it cannot be easily performed in a parallel fashion with multiple substrate concentrations. It is also necessary to exclude atmospheric carbon dioxide from the aqueous media containing the enzyme and substrate. 

Describe the two types of information that can be obtained about a reaction by using the pH-stat method of titration. 

Recently a new pH-stat instrument (PHM290 with autoburet ABU901) was introduced by Radiometer International A/S. This instrument can be connected directly to a personal computer, so that titration data and pH can be recorded in a Windows-based program. A recording of corrosion potential must then be made by another instrument, but both titration data and potential measurements can be recorded simultaneously using two inputs. 

A hydrolytic reaction that releases acid may be followed by titration with base. This is best done automatically by use of the pH-stat. A glass electrode registers the pH of the solution, which is kept constant by the automatic addition of base from a syringe controlled by an electronic circuit. Reaction volumes as low as 1 mL may be used, and the limit of detectability is about 50 nmol (5 to 10 /tL of base at 5 X 10-3 to 1 X 10-2 M). The usual source of error with this apparatus is the buffering effect of dissolved C02. 

The pH-stat technique monitors the course of a reaction in which each peptide bond is hydrolyzed by one or several enzymes. pH-stat evaluates the progress of hydrolysis by titrating the released amino groups with an alkaline solution. Enzymes work at constant pH and temperature during the entire process, so that no buffering is needed. 

A pH-stat or automatic titration instrument greatly simplifies titrimetric analyses in that no buffer is required and the time course of tritrant addition may be available as a continuous recording (facilitating estimates of initial rates see Critical Parameters). However, one limitation of the automatic titrator compared to the... 

Protein hydrolysis, evaluating degree of fluorescamine reaction, 146-147 formol titration, 147-148 ninhydrin reaction, 142-144, 153 pH-stat, 149-151, 153 TCA solubility index, 152 TNBS reaction, 144-146... 

A somewhat more elaborate assay has been proposed by Blakeley et al. (13) employing a recording pH stat. In this assay no buffer is used, but dithiothreitol (DTT) is present during the titration (that maintains the pH at 7.0). The unit, IU, is defined as the amount that causes the decomposition of 1 jumole of urea per minute (38°, pH 7.0, 0.05 M urea, 2 t M DTT). One Sumner unit = 17.6 IU/mg = 11 Uf/mg. Specific... 

Phelan and Mattigod (1987) studied calcium phosphate precipitation from stable supersaturated solutions using pH/Ca-stat and pH-stat. The pH and Ca2+ activity of the titrand were kept constant utilizing ion-specific electrodes attached to automatic titrators. A schematic diagram of the apparatus used by Phelan and Mattigod is shown in Fig. 3.2. 

Initial scale up of the enzymatic resolution for production of kilogram quantities of (R)-2-amino-2-ethylhexanoic acid was performed in a batch process. The oil of ethyl 2-amino-2-ethyl-hexanoate was suspended in an equal volume of water containing the enzyme. The enantioselective hydrolysis of the ester proceeded at room temperature with titration of the produced acid by NaOH through a pH stat (Figure 6.4). 

A related form of an automatic potentiometric titrator is instrumentation that permits the maintenance of the acidity or basicity of a solution over a period of time. Such devices are known as pH-stats, and find application in kinetic studies of hydrolysis reactions. The general approach is (by either manual or automatic means) to add either acid or base such that the pH in the solution is maintained constant over a period of time. Normally the amount of acid or base added as a function of time is sought in order that kinetic measurements may be made for the system. In its simplest form the acidity of the solution is monitored with a pH meter and controlled at a preselected value by the addition of acid or base from a burette the quantity delivered as a function of time is recorded in a notebook. Obviously for the fast reactions this becomes difficult and dependent on the dexterity of the individual. 

Both the automatic coulometric titration of petroleum streams and the continuous monitoring of pesticides and sulfur-halogen compounds indicate that the coulometric titrator method is amenable to the automatic maintenance of the concentration of a component in a solution system. A manual version of this approach has been used to study the kinetics of hydrogenation of olefins as well as to determine the rate of hydrolysis of esters.12 The latter system is a pH-stat that is based on the principles of coulometric titrations. Equations (4.9)-(4.11) indicate how this approach is applied to the evaluation of the rate constants for ester hydrolysis. A similar approach could be used to develop procedures for kinetic studies that involve most of the electrochemical intermediates summarized in Table 4.1. The coulometric titration method provides a convenient means to extend the range of systems that can be subjected to kinetic study in solution. 

Automatic Recording Titrimeter Use an instrument operating in the pH stat mode and equipped with a jacketed titration cell (Radiometer Titralab, or equivalent). 

Titration curve differences may be measured directly by means of a pH-stat (Jacobsen et al., 1957). We begin with the protein in its initial native state at a particular pH, and then allow denaturation, metal binding. 

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