Determination of the Relative Protein Concentrations of Cell Free and Stage 1 Extracts from the Purification of Alkaline Phosphatase.

Abstract In order to quantify total protein for any stage in the purification of an enzyme or protein, a rapid, sensitive, and reliable method for protein concentration determination is required. In an attempt to measure the relative concentration of the cell free and stage 1 extracts from a purification of E. coli Alkaline Phosphatase, the Bradford dye binding assay was used. A standard curve was developed using a series of Bovine Serum Albumin (BSA) standards in the 100 g/ml to 1,500 g/ml range. The absorbance of each sample was measured at 595 nm and plotted versus [BSA]. The resulting line was fit by the linear least squared method. Samples from the cell free and stage 1 extracts were subjected to the Bradford assay in the same fashion. In theory, the measured absorbance of each together with the equation for the line generated in the BSA standard curve should allow determination of the relative protein concentrations in each sample. The relative protein concentration for the cell free extract was determined to be 1,375 g/ml, however, the apparent non-linearity of the standard suggests that this number may be inaccurate. The relative protein concentration for stage 1 was determined to be negative suggesting that the sample measured was too dilute for the sensitivity range of the Bradford method. In order to address these problems so that protein concentrations can be reliably determined, the range of BSA standards used will be reduced to span 100-1,000 g/ml. In addition, a larger volume of stage 1 will be used in the assay in an attempt to provide sufficient protein with respect to the sensitivity of the method. Alternatively, the sample could be concentrated several fold, thereby allowing the lower sample volume to be used. The successful establishment of a reliable standard curve will greatly facilitate analysis of future protein preparations as well as provide quantitative measures of the purified enzyme that will be required for characterization of the enzyme.

Introduction Alkaline phosphatases (orthophosphoric monoester phsphohydrolases, EC 3.1.3.1) are phosphate hydrolases that catalyze the following general reaction at high pH (>7.0): R-O-PO3H- + H2O R-OH + H2PO4-

These enzymes perform important functions at the cellular level in a wide variety of organisms, both eukaryotic and prokaryotic. The physiological role of alkaline phosphatase in E. coli is to provide the cell with a source of inorganic phosphate (Pi). It accomplishes this task by cleaving phosphoryl groups from a wide variety of phosphorylated compounds. These reactions take place in the periplasmic space between the outer peptidoglycan membrane of gram-negative bacteria and the cytoplasmic membrane of the cell. The outer membrane is permeable to

many phosphorylated compounds while the cytoplasmic membrane is not. Alkaline phosphatase liberates Pi in this space where it becomes bound by the Pi-binding protein. This protein delivers phosphate to the high affinity Pst transport system, which transports phosphate across the cytoplasmic membrane (1). Because enzymes of this class perform such an important function in a wide variety of eukaryotic and prokaryotic cells, it is of considerable interest to isolate and characterize the physical properties of a representative member of this enzyme class. A thorough characterization of this essential enzyme will provide important insight into the catalytic event that provides inorganic phosphate to the bacterium. In addition, this study will provide a general knowledge base for the study of homologous alkaline phosphatases from eukaryotic sources. In an effort to provide the necessary foundation required for the complete characterization of the major alkaline phosphatase from the bacterium, Escherichia coli, purification of the enzyme is required. During the course of protein purification, it is often necessary to measure the concentration of various samples. Such information allows an evaluation of purity to be made as well as providing some idea of the amount of protein that is available for further use. Various methods for determining protein concentration in a given sample are available, but they vary in ease of use, sensitivity, and cost. We chose to use the Bradford method (2), which is a dye binding method. The dye, Coomassie brilliant blue G-250, has a maximum absorbance at 465 nm. When it binds to a protein under acidic conditions, the maximum shifts to 595 nm. The binding of the dye to protein involves strong noncovalent interactions including electrostatic, between amino and carboxyl groups, as well as van der Waals forces (1). Because all proteins possess amino and carboxyl groups and participate in both electrostatic and van der Waals interactions, this method is largely insensitive to protein structure at any level. The method can be used develop a standard curve for known concentrations of any abundant protein. The linear relationship between the absorbance and known protein concentration can then be used to determine the relative protein concentration of any given sample. In the current study, the Bradford dye will be mixed with known concentrations of a protein, in this case Bovine Serum Albumin (BSA). The absorbance of each standard will be measured at 595 nm. The straight-line relationship between absorbance and concentration will be fit by the least squares method. The concentration of two samples, cell free extract and stage 1 from an alkaline phosphatase purification, will be determined using the slope and intercept from the derived equation. Materials and Methods Preparation of Standards BSA was made purchased in crystalline form from Sigma Chemical (St. Louis, MO). 0.1 g of BSA was dissolved in 10 ml of water at room temperature.

The stock BSA solution was diluted to span the 100-1,500 g/ml range in the following manner:

Table 1. Standard BSA solution preparation [BSA] g/ml 100 200 400 600 900 1,200 1,500 Volume (L) of 10 mg/ml BSA Stock 5 10 20 30 45 60 75 Volume (L) of MilliQ water 495 490 480 470 455 440 425

60 L of each standard was mixed with 940 L of Bradford reagent. This was repeated twice for each concentration allowing three measurements to be made for each concentration of standard. Each sample was allowed to incubate at room temperature for 10 minutes and no longer than hour before being measured. The absorbance of each standard was measured at 595 nm against a blank that was composed of 60 L of water and 940 L of Bradford reagent. Absorbance was plotted against [BSA] and an equation for the line was generated. A sample from the cell extract and the first stage of a current and ongoing preparation of alkaline phosphatase was prepared and measured in the same manner as the standards. Three identical measurements were made for each sample.

Spectrophotometer Parameters Measurements were made on a Cary 1 dual beam spectrophotmeter that had been warmed up for at least hour prior to measurements. Measurements were made in the absorbance mode at 595 nm using a 1 second integration time. The instrument was zeroed by placing blanks in both the front and rear cavities and instructing the instrument to set the difference in absorbance equal to zero. While keeping the blank in the rear cavity, samples were placed in the front cavity and their absorbances were recorded.

Data/Results Table 2. BSA Standard Curve Absorbance Measurements [BSA] g/ml 100 200 400 600 900 1,200 1,500 A595 0.07 0.21 0.4 0.59 0.81 1.1 1.2 A595 0.08 0.19 0.41 0.6 0.79 1.05 1.15 A595 0.09 0.20 0.39 0.61 0.8 1.15 1.25 Average A595 0.08 0.20 0.4 0.6 0.8 1.1 1.2

Bovine Serum Albumin Standard Curve

1.4 Absorbance at 595 nm 1.2 1 0.8 0.6 0.4 0.2 0 0 500 1000 [BSA] ug/ml 1500 2000

Figure 1. In a standard reaction, 60 L of each BSA concentration was mixed with 940 l of Bradford reagent and allowed to stand at room temperature for ten minutes. Each sample was measured for absorbance at 595 nm against a blank, which contained 60 L of water mixed with 940 l of the Bradford reagent.

A linear least square fit of the line produced the following equation:

y = 0.0008 [BSA] + 0.05 Table 3. Absorbance of Protein Purification Samples Sample Cell Free Stage 1 Calculations Solving the equation for protein concentration using the cell free extract absorbance value: 1.15 = 0.0008(X) + 0.05 X = [BSA] = 1375 g/ml The same calculation for Stage 1 gives [BSA] = -25 g/ml A595 1.15 0.03 A595 1.1 0.01 A595 1.2 0.05 Average A595 1.15 0.03

Discussion The absorbance at 595 nm for several concentrations of BSA was measured and averaged. Table 2 clearly shows that little variation between measurements at low standard concentrations with respect to the change in average absorbance observed from standard to standard.was evident. This was not true for the two highest concentrations where the variation, 0.05 absorbance units, was on the order of the average change in absorbance from 1,200 g/ml to 1,500 g/ml. The data graphed in Figure 1 is not quite linear and the main reason for this appears to be the similarity in absorbance for the highest concentrations of BSA. Taken together, these observations indicate that the line derived from the standard curve could be made better by lowering the concentration range such that the absorbance change is linear with the change in concentration. Analysis of the two protein purification samples (Table 3) results in concentrations of 1375 g/ml for the cell free extract and 25 g/ml for stage 1. Although the absorbance and concentration of the cell free extract are reasonable with respect to the standard curve, it is likely not reliable as a measurement for two reasons. First, the line derived from the standard curve is non-linear as discussed above so the best estimation of concentration cannot be obtained from this relationship. Second, the absorbance observed for this sample is on the high end and likely suffers from the same problem as the two highest concentration standards, that is that their concentrations are sufficiently high to be out of the linear range of response for this method. To solve this problem, a dilution of the sample can be made before treating it with the Bradford reagent

so that the measured absorbance falls into the linear range. The stage 1 sample suffers from the opposite problem. Because it is not possible to have a negative concentration, there is clearly a problem with the measurement of Stage 1. Table 3 shows an absorbance (A595 = 0.03) for this sample that is lower than any of the absorbance values recorded for the range of BSA standards. This value is also close to the reliable detection limit (A = 0.001) of the spectrophotometer. Taken together, it appears likely that the sample is too dilute in protein concentration to be measured by this method. A likely solution to the problem is to concentrate the sample or possibly use a larger volume (>60 L) of the sample in the assay. In summary, the current method of protein concentration determination has resulted in questionable results. Therefore, reliable concentration values for the two protein purification samples cannot be reported. The method will be repeated with changes that include lowering the span of standard concentrations, diluting the cell free extract sample prior to assay, and finally concentrating or using a larger volume of the stage 1 sample in the assay.

References 1. Ninfa, A.J & Ballou, D.P. 1998. Fundamental Laboratory Approaches for Biochemistry and Molecular Biology, Fitzgerald Science Press, Inc., Bethesda, Maryland. 2. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248.