Phosphate serum buffering

Binding Constants (K) for Flavonoid-Serum Albumin Complexes (1 1 Stoichiometry), pH 7.4 Phosphate-NaCI Buffer, 25°C... 

A normal adult has a total body phosphorus content of 700-800 g [1]. The majority of phosphate is present in bone, although approximately 15% is distributed outside of the skeleton where it is present in the form of inorganic phosphate in extra-cellular fluid and organic phosphates within cells, such as adenosine triphosphate (ATP), nucleic acids, and membrane phospholipids. As such, phosphorus plays a vital role in numerous cell processes including cell energetics, cell membrane formation, and DNA RNA synthesis, to name a few. Within blood, phosphate exists mainly in two forms, HPO and HjPO. These two anions are important serum buffers and their relative concentrations are determined by the serum pH. 

Fig, 3.32 exhibits the normalized fluorescence emission spectra of calcofluor free in phosphate-NaCl buffer (b), in the presence of a saturated concentration of a i-acid glycoprotein (c) and of HSA at a concentration close to saturation (a). The emission maximum of the fluorophore shifts from 435 nm in buffer to 448 or to 415 nm, respectively, in the presence of a i-acid glycoprotein or HSA. The red shift (13 nm) of the maximum observed in presence of a i-acid glycoprotein compared with the maximum obtained in water indicates that the microenvironment of the excited state of calcofluor on a i-acid glycoprotein is hydrophilic. The blue shift (20 nm) observed in presence of the serum albumin corresponds to an emission from a hydrophobic environment. Thus, the interaction of calcofluor with serum albumin arises from molecular energy transitions different from those present in the interaction between the fluorophore and a i-acid glycoprotein. 

Unadsorbed serum from previous step was put a second time on the same immunoadsorbent, washed with phosphate/saline buffer pH 7.2, and eluted with 5 M guanidine HCl then the above procedure was followed. 

FromSutherlandeta/. (2002). Oliveoil (67.3% 18 1,12.4% 18 2)and safflower oil (12.7% 18 1,77.6% 18 2) were heated in a deep-fryer for 8 hr at 210°C, with potato chips added after every hr. The heated and unheated oils were used in ice-cream milk shakes and fed to 16 healthy men. The serum separated from venous blood was diluted (0.67%) in phosphate-saline buffer, oxidized with copper (12.5 Ttmol/l) at 25°C and the formation of conjugated dienes was followed for 4 hr at 234 nm. 

Three different types of columns packed with gels of different pore sizes are available. Columns should be selected that are suitable for the molecular weight range of specific samples, as each type has a different exclusion limit (Fig. 6.41, page 215). Bovine serum albumin (BSA), myoglobin, and lysozyme show good peak shapes using only 100 mM of sodium phosphate buffer as an eluent. There is no need to add any salt to the eluent to reduce the ionic interaction between protein and gel. 

FIGURE 7.10 Dependence of the resolution on the sample volume. A preparative Superformance column 1000-200 (bed volume 20 liters) packed with Fractogel END BioSEC (S) (bed height 63 cm) was loaded with 60 ml (top) and 300 ml of a mixture of bovine serum albumin (5 mg/ml), ovalbumin (5 mg/ml), and cytochrome c (3 mg/ml) (bottom) (20 m/VI sodium phosphate buffer, 0.3 M NaCI, pH 7.2 flow rate 100 ml/min corresponding to 19 cm/hr). When the sample volume is 300 ml the separation efficiency for BSA and ovalbumin is similar. Thus the column can be loaded with larger sample volumes, resulting in reasonable separations. 

MCPBA converts the MTM ester to a methylsulfonylmethyl ester (78-98% yield), which can be hydrolyzed enzymatically with rabbit serum (pH 4.5 phosphate buffer, EtOH, 25-28°, 1 h, 84% yield). ... 

Calf kidneys, dog kidneys and rhesus monkey kidneys were treated with trypsin to give suspensions of cells. The suspensions were centrifuged and the packed cells diluted with 400 volumes (calf cells) or 200 volumes (dog cells and rhesus monkey cells) of a growth medium consisting of 5% horse serum and 0.5% lactalbumen hydrolysate in Earle s saline, with 100 units/ml each of penicillin and streptomycin. These media were used separately to produce Semliki Forest/calf interferon, Semliki Forest/dog interferon and Semliki Forest/rhesus monkey interferon. The cell-containing growth medium was dispensed into 500 ml medical flat bottles (70 ml in each). The cultures were incubated at 36°C. Confluent sheets of cells (monolayers) were formed in 5 to 6 days. The growth medium was then removed and the monolayers were washed with isotonic phosphate-buffered saline, pH 7.5. 

FIGURE 9.6 DSC of (a) recombinant resilin in water showing no enthalpic events, (b) bovine serum albumin in phosphate-buffered saline (PBS) showing denaturing occurring at 62°C, and (c) wool fiber in water showing denaturing of the a-helix at 145°C (Endotherm up). 

In the Phadebas TM amylase test (72) (Pharmacia Labs) the substrate was a water insoluble cross-TTnked blue starch in tablet form which also contains some inert ingredients, sodium and potassium phosphate buffer salts and sodium chloride. This polymer was hydrolyzed by amylase into water soluble blue starch fragments. After centrifugation the absorbance of the blue supernatant was proportional to the activity of amylase present in the test samples. The day to day variation on a quality control serum had a coefficient of variation of 2.7% based on 30 days of data in our laboratory. The method is simple, reproducible and uses microquantities of serum. 

Figure 5. Fluorescence anisotropy of F-D labelled heparin-antithrombin interaction. F-D-heparin (0.02 fluoresceins per uronic acid) at 0.1 mg/ml was incubated with different concentrations of antithrombin (open circles) or bovine serum albumin (solid diamonds) in 20 mM sodium phosphate buffer, pH 7.4.

Preparations of PEG-modified proteins. A. SC-PEG (1 g, 0.2 mmol) was added to a stirred solution of Bovine Serum Albumin (BSA) (100 mg, 1.5 x 10 6 mol) in 0.1 M sodium phosphate, pH 7.8 (60 mL). Sodium hydroxide (0.5 N) was used to maintain pH 7.8 for 30 min. The excess of free PEG was removed by diafiltration using 50 mM phosphate buffered saline. Approximately 30 amino groups of the native protein were modified as determined by trinitrobenzenesulfonate (TNBS) assay (28). The same degree of modification was obtained when the experiment was repeated under identical conditions using SS-PEG instead of SC-PEG. 

Fig. 10 Release of cromolyn sodium (sodium cromoglycate) from human serum albumin microspheres prepared using a water-oil emulsion technique with 5% glutaraldehyde as cross-linking agent. Dissolution medium pH 7 phosphate buffer. (From Ref. 98.)... 

Wakabayashi et al. [51] determined penicillamine in serum by HPLC. Serum (0.1 mL) was vortex-mixed for 30 s with 50 pL of 0.1% EDTA and 0.2 mL of 10% TCA. The solution was centrifuged at 1500 x g and filtered. A 5 pL portion was analyzed on a Shodex C18 column (15 cm x 4.6 mm i.d.), using a mobile phase of 19 1 methanolic 0.05 M phosphate buffer (pH 2.8) containing 1 mM sodium octylsulfate and 10 pM EDTA. Liver or kidney samples were similarly extracted, and the extracts were cleaned up on a Bond-Elut cartridge prior to HPLC analysis. Detection was effected with an Eicom WE-3G graphite electrode maintained at +0.9 V versus Ag/AgCl. The calibration graph was linear up to 500 ng, and the detection limits were 20 pg. For 1 pg of penicillamine added to serum, liver, or kidney, the respective relative standard deviations (n = 5) were 3.6, 5.1, and 4.4%. 

C for 1 h. A 100 pL portion of the solution was injected onto a column (15 cmx 3.2 mm) of LiChroscob RP-18 (7 pm) for HPLC at room temperature, using acetonitrile-0.033 M phosphate buffer of pH 8.2 (1 2) containing 0.05% of ethyle-nediamine as the mobile phase (eluted at 1 mL/min). Fluorimetric detection involved excitation at 338 nm and measurement at 540 nm (or with a 430 nm cutoff filter). For 50 300 ng of drug injected on to the column, the coefficient of variation was 7-8%. The method permits a simple determination of (z>)-penicilla-mine in serum at therapeutic levels. 

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

Figure 3. Autoradiograph of radio-iodinated bovine serum albumin (BSA), adsorbed onto glass and subsequently immersed in phosphate-buffered saline for (from left to right) 0, 1, 2, 4, and 16 h. Reproduced from Michaeli et al. (7) with permission of Academic Press.

After initial cell fermentation and product extraction from the producer cells, the crude preparation is subject to multiple chromatographic steps, including ion-exchange, hydrophobic interaction chromatography and gel-filtration chromatography. The purified product is presented in lyophilized form in vials (1 mg active/vial) and excipients include a phosphate buffer, sodium chloride and serum albumin. 

The protein A (pA), antihuman serum albumin (a-HSA, M 150 kD), and human serum albumin (HSA, M 65 kD) were provided by Paradocs BV (Tiel, The Netherlands). The Herpes Simplex Virus type 1 (HSV-1) and anti-HSV-1 gG glycoprotein G monoclonal antibody (a-HSV-1 gG) were purchased from Virusys Corporation (Marriottsville, MD, USA). Bovine serum albumin (BSA, M 50 kD) was purchased from Sigma-Aldrich Chemie BV (Zwijndrecht, The Netherlands). Synthetic surface protein of Hepatitis-B virus generated in Hep-G2 cell-line (HEP G2, M 25 kD) was provided by BioMerieux BV (Boxtel, The Netherlands). Phosphate buffered saline (PBS) was used for all experiments. 

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