Manipulation of phosphate from organic phosphates present in acropolises during seed germination and seedling growth are largely carried out by acid phosphates.
(Duff et. Al. , 1994) In the purification of enzymes, cells must first be disrupted and selectively purified. These steps must be undertaken without a resultant degradation of the enzyme and loss of enzyme activity. Plant acid phosphates are found predominantly in superheroes in the cell.
Many plant acid phosphates occur in small quantities with high instability in dilute solutions.These factors, along with the fact that plant acid phosphates tend to occur in multiple arms makes the isolation of highly purified acid phosphates increase the chance of denomination during purification. .Growth of seedlings during germination and functioning of metabolic processes are largely dependent on phosphates catalysts solicitation and manipulation of organic sulfates in soils (Bison et. Al. , 1993) There are many variations in isomers within species.
Chromatographic methods have facilitated purification of individual isomers. It is important to use a standard assay for all activity and concentration measurements.In this paper, the isolation, partial reification and characterization of maize acid phosphates from maize germ are reported. Experimental Procedures Sample preparation Maize grains of mass 45 g were steeped in water for 48 hours.
The water was changed occasionally and placed between two damp cloths and allowed to stand until the maize germinated. Initial Extraction of Acid Phosphates All fractionation processes were carried out at COO. Seeds which had not germinated were discarded and 40 g of the remainder was blended with an electrical blender.Ice cold water of volume mall was added to the crude and the containing vessel as allowed to stand on ice for 30 minutes with stirring. It was then strained with a clean cloth and centrifuged at 10000g for 20 minutes at COO and filtered through Whitman No. 1 filter paper.
Triton-X 100 of volume 6 ml was added and the sample was divided into two portions. One portion was stored in the fridge and the other sample was stored in the freezer at a temperature of -ICC. Determination of Protein Concentration by the Folio-Lowry Assay To 1 ml of phosphates solution in a test tube, 5 ml of alkaline reagent was added and mixed.This was left to stand for more than 10 minutes.
To the sample in the test tube, 5 ml of Folio-Cacciatore reagent (diluted 1 in 3) was added with rapid mixing. The absorbency was read after 30 minutes at a wavelength of 750 NM. The protein concentration was determined using 0. 1% bovine serum albumin (BAS) with a concentrations ranging between O and 1 MGM/ml.
Specific Activity Assay A tube containing 2. 0 ml DEED-citrate buffer of pH 5. 0 and 2. 0 ml of 2.
5 mm of p- interruption was prepared. The tube was placed in water bath of temperature ICC for at least 10 min for it to equilibrate.Aliquots were diluted with 0. 1% BAS to appropriate concentrations. To the reaction mixture, 1 ml of enzyme was added with an immediate transfer of 0.
5 ml of the reaction mixture into 5. 5 ml of 0. 1 M Noah to quench the reaction. The calibration curve used for the determination of p- interruption amount was constructed using six test tubes containing known amounts of p-interruption. Six tubes containing 0.
0 to 5. 0 ml of 60 PM p-interruption solution with intervals of 1 ml were set up. Each tube was brought to a total volume of 2 ml by adding 0. 02 M Noah.The enzyme activity was determined using Figure 42. The estimated mol of p-interruption produced in 30 min was 0.
742 mol. The activity of the enzyme in the freshly prepared extract was 0. 257 units/ml. Figure [4] [2] Calibration Curve used to Determine Enzyme Activity of the Fresh Crude Sample Crude Thawed after a Week of Freezing A sample which was kept frozen for a week was thawed out and assayed in order to establish the effect freezing had on the enzyme activity and concentration. The activity was determined using the assay in section 3.
4 and the corresponding protein concentration was determined using the Folio-Lowry assay.Figure and Figure 44 were used to determine the concentration and enzyme activity of these samples. The concentration obtained was 8. 66 MGM/ml and the enzyme activity was 0. 292 mol/min.
The specific activity of the sample was 0. 0337 mol/min. Figure [4] [3] Calibration curve for the determination of the concentration of protein concentration of samples thawed out after a week of freezing. The curve was also used to determine the protein concentration of the samples eluted on the Sapheads 25 gel. Figure [4] [4] Calibration curve for the determination of enzyme activity of samples thawed out after a week of freezing.
