Utilizing Acetone Powder of Several Organs to Compare Specific Proteins and Measure L-Lactate Dehydrogenase Activity Abstract Animal organs contain a diverse amount of proteins that define their function. In this study, acetone powder solutions of calf liver, bovine pancreas, chicken muscle, porcine kidney, porcine brain, and porcine heart were analyzed through an SDS-Page gel to determine diversity of proteins and through an LDH assay to determine enzymatic activity. It was found that chicken breast muscle contained the most diverse collection of proteins along with the highest LDH activity. This made reasonable sense considering that the chicken breast muscle is composed of many different classes of actin and myosin proteins and that …show more content…
Measuring Protein Concentration of Extract Using the Bradford Method Protein concentration needed to be determined to assure that SDS-PAGE would work effectively since too high or too low protein concentration of each sample would result in erroneous gels. Before measuring the protein concentration in the acetone powder solutions, a BSA curve needed to be created to find a conversion factor. In the BSA curve, there appeared to be a positive correlation between absorbance (A) and the amount of BSA protein (μg) when observing the best fit line (Figure 1). This was confirmed with a slope or conversion factor of 0.0255 A595/μg (Figure 1). To determine protein concentration in the acetone powder suspensions, three representative samples in the range of the BSA standard curve were chosen to determine the amount of protein in each solution. The largest protein concentration was found in the bovine pancreas tissue (6.130 ± 1.05418 mg/mL), while the calf liver had the smallest protein concentration (0.003 mg/mL) (Table 1). The chicken breast muscle solution had the largest concentration of protein (2.335 mg/mL) after the solutions were adjusted for gel loading (Table …show more content…
The chicken breast muscle had the highest number of proteins present (13), while the porcine kidney had the least (3) amount present (Table 2). The protein with the largest mass appeared to be the first protein in the chicken breast muscle lane at 192.5 kDa (Table 2). There was also a trend that two protein bands with an apparent mass of 64.6 ± 2.67 kDa and 78.5 ± 2.13 kDa appeared in each of the different acetone powder lanes (Table 2). The protein with the smallest mass was found in the calf liver at 17.0 kDa (Table
Gelatin is observed to have a lower absorbance reading than lysozyme in only the Bradford assay, while in the BCA assay gelatin was observed to have about the same absorbance as lysozyme. In Bradford assay, the color yield for proteins with higher content of tryptophan, tyrosine, or cysteine residue, will be higher. The observed protein sensitivities did not correlates with the amino acid content in the Bradford assay. As presented in Table I, the mole fractions of lysine and arginine in BSA were 10 mole percent and 4 mole percent, respectively. The sensitivity should have been higher in BSA than in the Bradford assy. However, the observed sensitivities did correlates with the amino acid contents in the BCA assay.
The purpose of this study was to compare contractility of individual preparations glycerinated rabbit psoas muscle exposed to two different solutions known to cause muscle contraction. A standard contracting solution without 15 mM of creatine phosphate and a second contracting solution with 15 mM of creatine phosphate were compared to one another. Creatine phosphate has been found as
10.State a commercial use for biochemical testing as performed in this online laboratory. (5 points)
The normalized SDH activity of two homogenates can be compared by looking at the class statistics for the Liver and Kidney homogenate samples in the data sheet attached. The kidney exhibited higher enzyme SDH activity than the liver. This was in agreement with the proposed hypothesis. Comparing the same two homogenates in which malonate was present, it can be seen that the kidney exhibited higher SDH activity than the liver. Thus, both homogenates did in fact have a decrease in enzyme activity, as malonate inhibits the activity of SDH. In successive experiments more malonate was used, and class statistics, not the activity itself, showed lower amounts of enzyme activity/mg protein as reaction number increased and a greater significance. Thus, malonate’s effect did increase proportionally to its concentration. There was a significant difference in SDH activity between the liver and kidney homogenates (p=0.0001)0.05). (Figure 1)
Enzymes mediate almost every single biochemical reaction, process or metabolic event in the body. Overall, enzymes are proteins whose primary function is to catalyze, increase the rate of the biochemical reactions (Champe et al., 2005). Enzymes are not only efficient in increasing the rate or velocity in a biological reaction, but also are incredibly accurate at recognizing other biochemical structures to create specific products. Taking into consideration how diverse the properties of enzymes are and their importance in the biological process of the human body, it is not a surprise how enzymes can be used as invaluable tools in the diagnosis of certain diseases, therapeutic applications and innumerable cases of clinical trials and lab analysis (Devlin, 2011). One example of the many application of enzymes in the medical field is how Proteases Pronase (Hydrolytic enzyme) and RNases are being used to remove adhesions in contaminated
Once labeled, 2.0 ml of distilled water was placed into tube A and 2.0 ml of pancreatic extract was placed in tube B. Stock liquid protein was obtained and using a graduated cylinder, 5 ml of the liquid protein was placed in each tube. The tubes were then covered with Parafilm and inverted to mix the liquids. Both tubes were placed in a 37°C water bath for 15 minutes. After the 15 minutes, the tubes were removed from the water bath and placed in an ice bath for 10 minutes. Once the 10 minutes were over, the tubes were removed from the ice bath and observations were recorded. A ninhydrin test was performed on the amino acids by placing a drop from each tube on a piece of filter paper. The drops were circled and labeled A and B. A drop of ninhydrin was added to each spot and was dried with a hair dryer to heat-treat the piece of filter paper. The results were recorded in a
The concentration of bound protein was found to be 67.1g per 100g of protein powder. Each serving also contains 5g of natural sugar. The product does not contain any artificial ingredients or sweeteners. Also present are over 5g of BCAAs.
Accuracy and Precision were obtained by measuring the concentration of three variations of a simulated sample created by mixing SV, ASP and SAC then dissolved in a solvent mixture of methanol: water (60: 40). Simvastatin's concentration were (0.6; 1; 1.5 μg/ml), whereas the Aspartame's and Saccharin's concentrations were fixed at 0.5 and 0.3μg/ml. Each variation concentration was measured on SV wavelength measurement respectively in six times and then calculated %recovery and relative standard deviation %RSD. Intraday and interday precision were obtained from the determination of the SV in co-crystal form with claimed concentration 0.8 μg/ml in two
Chemicals were purchased from Aldrich chem. Co. England. Fresh human serum (obtained from Medical Research Institute Blood Bank) was used as source of the enzymes. The enzymes SALT and SAST activities were assayed by the method of Reitman and Frankel.(13) In this method; the keto acid formed were measured colourimetricaly after combination with 2,4-dinitrophenyl hydrazine, then the hydrazone formed was measured at 530
These are components of muscle tissue and aid in muscle growth. The overall main active ingredient to be concerned with is the whey protein isolate itself. A study done by Michaela C. Devries and Stuart M. Phillips on muscle Protein synthesis shows that “Whey protein is the optimal protein source to support MPS (muscle protein synthesis) at rest and following resistance exercise as well as to induce muscle hypertrophy and strength gains with resistance training”
While resting, the rabbit’s muscles were relaxed, and the acetyl-CoA was limited so reactions were
Blood serum contains a large amount of protein, and albumin is the main protein found in the blood, beside a large of other proteins are globulins. Basically albumin is synthesized as preproprotein in the liver by hepatocytes at a rate of approximately 12 grams per day, which constitutes about 25% of the total production of proteins in the liver. As well as after an average life of between 17-20 days the dismantling of the large part of albumin are in the liver too, most of albumin (about 60% of it) in the body fluids outside of the blood vessels, while the presence of the remaining 40% in blood serum.
Michaelis-Menten graphs depicted by Figure 4 and Figure 5 show that all concentrations of oxalic acid (0M, 0.01M and 0.03M) and oxamic acid (0M, 0.0200M and 0.0600M) ) seemingly saturate at the highest lactate concentration which was 95mM. In both figures, LDH saturates the fastest in the absence of inhibitors, followed by 0.01M oxalic acid and 0.0200M oxamic acid, and 0.03M oxalic acid and 0.0600M oxamic acid having the slowest reaction rate respectively shown in Figure 4 and Figure 5. These results reflect the Michaelis-Menten graphs reproduced by experiments under similar conditions as explained by Powers et al [9].
For day two of the experiment, the peptide containing digestion solution was submitted to the URMC Proteomics Resource Laboratory for liquid chromatographic separation and
L-lactate dehydrogenase, or LDH, is an oxidoreductase that occurs in many organism and is a reaction that is important to many cells. From the peptide sequence, this specific enzyme catalyzes the pyruvate to lactate reaction along with the coenzyme NAD+/ NADH. The enzyme classification for this sequence is EC: 1.1.1.27, these numbers each identify a specific part of the enzyme and the reaction is a part of. The first one identifies that this is and oxidoreductase reaction, which accounts for the dehydrogenase in the name. The second number is the labeling that a hydrogen is undergoing the oxidation reduction and the final one is indicating that the l-lactate is the acceptor of the hydrogen. The final digit, 27, is specific to the pyruvate reaction that the L-lactate dehydrogenase is catalyzing. Each peptide sequence was run through BLAST technology both forward and backwards. The sequence was examined multiples ways to understand that the order of a sequence is important, and to guarantee the correct enzyme is found. The BLAST database compares the sequence to many different organism and their different cellular reaction and rates each enzyme on their similarities and the likelihood of getting a certain result of a different database (E-value). The LDH was the top 100% match with a low E-value of 2e-11. This connected the sequence to an enzyme in the organism Mus Musculus in the muscle and heart cells. It occurs in different regions of the house