Human Carbonic Anhydrase II

Human Carbonic Anhydrase IIHuman carboniferous anhydrase II is one of the prompt studied enzymes cognize with a variety of percentages in reply catalysis. Its native function is to catalyze the reversible hydration answer of carbon dioxide. In addition to carbon dioxide hydration, it is similarly adequate to(p) of early(a) latent skills, such as catalyzing esterase activity. The ability of human carbonic anhydrase II to function as a catalyst derives from key residues in and around the bustling site that play of the essence(p) roles in the mechanism. Substitutions to two of those particular key amino acids were performed via Quick-change site directed muta agentsis H64A and V142D, to investigate the particular role they bring in the catalytic energetic site. Various kinetic experiments and structural analyses were performed on wild-type carbonic anhydrase and the mutants to fleck and compare their activity to each other and to literature, including Michaelis-Menten pa rameters for PNPA hydrolysis, carbonic acid gas hydration, and inferring function molecular(a) manikin. Though the same trends stick come in be seen as the literature, individual values were found to be much unkepter owing to errors in measurement and equipment. Trends were found to coincide with the mutants known roles in the active site His64 is the proton shuttle that facilitates proton transfer during the outrank throttle step and Val142 participates in the hydrophobic pocket to bind and recruit substrates to interact with the active site. Mutations to twain of these sites show that enzyme efficiency and activity strongly decreases.IntroductionHuman carbonic anhydrase II (hCAII) is a surface metalloenzyme that catalyzes the fol ruggeding reversible reaction . The enzyme commonly functions to help shuttle carbon dioxide in red split cells to rid the body of metabolic waste, and catalyzes the hydrolysis of many aromatic esters 1, 2. Structurally, a coat ion is located in the active site, coordinated to 3 histidine residues (H94, H96, H119) and normally a hydroxide ion or water grain 2.The mechanism of hCAII retort through two major steps 1) the conversion of carbon dioxide to bicarbonate, and 2) the regeneration of Zn-OH by proton transfer. The active hydroxide that is bound to zinc nucleophilically attacks a virtuallyby carbon dioxide molecule, resulting in a bicarbonate ion fertilization to zinc 3. The zinc-oxygen bond breaks to afterward release a bicarbonate ion, which is replaced with water 3. The ZnOH bond is regenerated by a proton transfer to the external buffer, which is facilitated by the His64 residue 3. The proton transfer step is the rate limiting step of the reaction 3. The diazole show upion chain on the histidine residue is what gives it the ability to be a proton acceptor and donor. Mutations in that position (His64) usually result in decreased enzyme activity due to a lack of proton transfer however the reaction does pro ceed to a lesser degree without an active His residue, possibly due to its extensive water profits in the active site forming secondary proton wires 4.Carbonic anhydrase catalyzes one of the most rapid reactions it is one of the fastest enzymes studied 1. Its reaction speed is due, in part, by the amphiphilic nature of the active site 1. The hydrophobic side is use to bind carbon dioxide, age the hydrophilic patch functions to optimally bes summit the carbon dioxide molecule for the reaction 1. The hydrophobic wall forms a well-defined pocket near the zinc-hydroxide and is composed of the hounding amino acids Val142, Val121, Leu197 and Trp208. The hydrophilic patch consists of Thr198 and Glu106, which form a hydrogen bond network with the ZnOH to stabilize and orient it for nucleophilic attack on carbon dioxide 2. Therefore, any modifications to the hydrophobic pocket would change its structure, and consequently, its catalytic efficiency 1.In this study, the importance and role of His64 and Val142 to the structure and mechanism of hCAII are inflexible through site-mutagenesis and subsequent characterization of the fresh mutants, H64A (His64 Ala) and V142D (Val142 Asp) via kinetic and structural analysis. The changes that arise from the substitutions whitethorn prove to be applicable to drug synthesis because hCAII is known to be involved in a variety of diseases, for example, Marble brain disease, where genetic mutations in the hCAII gene leads to a deficiency in the enzyme which is an autosomal recessive disease 5. Studies in hCAII mutations can be utilise to formula folding modulators to suppress misfolding which frequently occurs due to hCAII destabilization 5. Another major disease involved with hCAII gene is osteopetrosis. The hCAII genes inactivation decreases osteoclast function in attire, and knowledge of hCAII mutations that inactivate the enzyme may lead to better chthonicstanding of bone remodelling 6. Some carbonic anhydrase diseases u se inhibitors (CAI) to suppress the hCAII as a therapeutic treatment. Inhibitors prevent hCAII activity by inhibiting either of the reaction steps the conversion of CO2 which involves V142 in the hydrophobic pocket, or the rate limiting step, proton transfer, in which His64 is crucial.Experimental ProcedureSite directed mutagenesis via the PCR-based Quick-change method was performed on hCAII as cited in Woolley (2011) for 10 ng and 20 ng wild-type plasmids (hCA2pET24b from Novagen) 7. put off shows the rate of the primers utilize in the PCR reactions. Products of PCR mutagenesis reactions were enumeration on 0.7% agrose gels to specialize size. The gels were run at 150 V in 1X TAE buffer. Red safe dye from intron Biotechnology was used in the agrose gel instead of ethidium bromide for safety fences 7. The standard molecular cant ruler used was a 1 kB desoxyribonucleic acid ladder from Fermentas.Table Primer sequences used in mutagenesis of hCAII in the precedent and lift cathexis for mutants H64A and V142DsportsmanDirectionSequenceMW (Da)%GCTM (C)H64AForwardGGATCCTCAACAATGGTgcTGCTTTCAACGTGGAG107785167ReverseCTCCACGTTGAAAGCAgcACCATTGTTGAGGATCC10709V142DForwardCTGATGGACTGGCCGaTCTAGGTATTTTTTTG98684462ReverseCAAAAAAATACCTAGAtCGGCCAGTCCATCAG9779The enzyme, DpnI, was then used to digest methylated deoxyribonucleic acid (the parent guidebook desoxyribonucleic acid). The desoxyribonucleic acid vector that dribbleed the mutation was transformed into supercompetent E.coli turbo cells from juvenile England Biolabs by heat shock 7. LB-agar plates were prepared to grow the transformed cells necessitateing mutant genes (i.e. H64A and V142D hCAII gene) 7. twain were injected with Kanamycin to run across that the culture that grows will capture the desired mutation 7. A miniprep culture was set-up from the LB-agar plate into LB median(a) to grow one colony for DNA analysis 7.Restriction enzyme mapping was prepared and XhoI and BglII were chosen, they wer e used under buffer 3 for optimal efficiency. Plasmid putification was performed victimization the QIAprep Spin Miniprep Kit, and then the chosen labour enzymes were carried out and were run on 1% agrose gel 7.A exemplar of the purified DNA was sent to an external follow (ACGT) for commercial sequencing (Sanger dideoxy type) to verify if the mutagenesis occurred correctly. The sequence was analyzed exploitation the program BioEdit. To happen the level of confidence of the sequencing results, the purified DNA was quantified using UV/Vis absorption via a spectrometer 7. The flirt withness was calculated using Ec1(260) as 50 g/mL.Purified plasmid DNA was transformed into E.coli BL2(DE3) cells to initiate protein expression by heat shock, mistakable to the transformation into turbo cells 7. The cells were cultured and a single colony was grown. Once sufficiently grown, ITPG and ZnSO4 were added to induce protein expression 7. SDS-PAGE was used to confirm protein expression and w as analyzed against an unvarnished protein molecular weight marker by Fermentas. The protein and ladder was stained with coo majorityie blue 7.Affinity chromatography was used to purify the mutant hCAII proteins 7. The hyaloplasm used was agrose linked to p-(aminomethyl)benzenesulfonamide, exploiting the tight binding that occurs amidst hCAII and sulphonamides. Once purified, the protein was dialyzed using a 6000-8000 Da dialysis membrane to replace the elution buffer with protein buffer and removes the matrix from the protein 7.SDS-PAGE is again used to confirm the protein is still present after purification and to check its approximate molecular weight. It was run for two different amounts of protein, 2 g and 10 g, and also ran 10L of wash fractions from affinity chromatography 7. Protein soaking up was determined by UV absorption at 280 nm in a final concentration of 6M guanidine hydrochloride. From the calculated concentrations, duty of the protein could be assessed via SD S-PAGE.To characterize this purified hCAII protein, a variety of analyses were done. Two types of battalion spectrometry (MS) were performed electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) 7. The MS analysis was used to confirm the presence of the mutation in hCAII with whole and digested protein. Protein samples (H64A and V142D hCAII) were not diluted for either of the MS analyses as cited in Woolley (2010). Samples of 10 L of stock protein concentrations (37.6 M H64A and 3.2 M V142D hCAII) were used for analysis of the molecular weight of the intact protein by ESI-MS. Both mutants were then digested by Trypsin Gold (MS grade) from Promega and the resulting fragments were evaluated by ESI-MS as well 7. A 50 L sample was used for each mutant, 40 L of the mutant at stock concentration and 10 L of the Trypsin Gold. A couple L of the digested mutants were saved for MALDI-MS and the rest was used for ESI-MS. Once the molecular weights for each of the digested fragments were determined by ESI-MS, the products were run through a protein database to confirm the identity of the protein and mutations 7.The 1 L of the tryptically digested mutants prepared for ESI-MS, subsequently underwent MALDI-MS. The 1 L samples were mixed with a matrix consisting of 1 L -cyano-4-hydroxycinnamic acid (CHCA) and 1 L of 0.1% trifluoroacetic acid (TFA) 7. The entire mixture was pipette onto a MALDI well and was inserted into the mass spectrometer and a MALDI-MS spectra was obtained.Michaelis-Menten kinetics was used to determine the KM and kcat of the p-nitrophenyl (PNPA) hydrolysis reaction 7. The ionized product from the hydrolysis, p-nitrophenol (PNP) produces a bright yellow twist that was used to follow the rate of the reaction via the Perkin Elmer Lambda UV/Vis spectrophotometer 7. Various sample concentrations of PNPA were set up to have a final enzyme concentration of 0.2 M in protein buffer 7. The sign rate measurements of each PNPA c oncentration were taken for wild-type enzyme, H64A mutant, V142D mutant, and a blank with no additional enzyme added (refer to data dodges in Enzyme dynamics I 7). PNP has a molar absorption coefficient () of 1.73-104 M1cm1. This was used to calculate Michaelis-Menten values Vmax, KM, kcat, and kcat/KM 7.The ability of hCAII mutants (H64A and V142D) and wild-type hCAII to catalyze the hydration of CO2 was measured. The pH of the solution was measured to track the progress of the reaction because the reaction generates protons. Enzyme solutions were prepared harmonize to table 2 in 7. The buffer used in the table was 50 mM TRIS buffer (pH 7.8). excess enzyme samples were prepared for 25 nM of wild-type hCAII and 100 nM of H64A mutant in a final concentration of 22.5 and 29.92 mM iminazole buffer (pH 7.8) respectively to determine chemical rescue of mutant H64A. The pH of the CO2 hydration taste was measured using a pH probe and pH meter at 5 second increments for a total of 90 seco nds starting at the beginning of the reaction 7. The slope of the sign changes in the first 2 points was considered to be the V0 for each enzyme concentration. From the initial swiftness, a kcat value can be calculated for each enzyme using the assumption that S KM, the Michaelis-Menten equation simplifies to kcat=V0/E.The third kinetics experiment used fluorescence to determine the binding constant of dansyl amide (DNSA) and acetazolamide (AZ) (from Sigma-Aldrich) to H64A and wild-type hCAII was performed using the Perkin Elmer Fluorometer 7. Stocks of 1 mM and 200 M of DNSA were prepared from a 21.6 mM DNSA stock by dilution with DMSO. Enzyme stocks were diluted to 0.25 M with TRIS buffer to make a 10 mL solution. A 1 mL sample of H64A from stock made was titrated with DNSA in small increments 7. The fluorometer emissions were taken at 470 nm. AZ titration in competition with DNSA was not able to be completed.The last characterization experiment done was molecularly modelling t he hCAII wild-type enzyme, as well as the mutants H64A and V142D. The molecular model of hCAII analyzed was derived by x-ray crystallography and found in the Protein Data Bank (PDB) repository. The wild-type and H64A hCAII structures examined had a PDB code of 1CA2 and 1MOO respectively. At present, no crystal structure has been found for V142D hCAII. The Swiss PDB Viewer program was used to visualize the protein structures. Secondary structures of the proteins were able to be ascertained. Residues around the metal active site and the Ramachandran plot were explored. Homology between hCAII and other carbonic anhydrase isozymes, hCAIV (PDB code 1ZNC) and hCAI (PBD code H1CB), were also studied by performing an iterative magic fit on the -carbons and structure alignment for each pair. The root mean square (RMS) between hCAII and the other isozymes were also analyzed to determine conserved and deviated regions in the structures. The binding of carbon monoxide gas in the hCAII active si te was also investigated (PDB code 3KOI). The structural inhibition of hCAII by AZ was also gleaned by structural analysis (PDB code 3HS4). Its mode of inhibition and binding sites were shown through the crystal structure. Lastly, the Swiss PDB Viewer program was used as a similarlyl to theoretically synthesize mutations and compare it to the veritable structure as determined by other scientists, for example, by aligning the virtual and crystallized mutations to determine deviations in structure by performing RMS.ResultsSite-directed mutagenesis PCR. Products from the PCR mutagenesis reactions were examined using 0.7% agrose gel electrophoresis. Two samples of differing amounts of template DNA (10 ng and 20 ng) were used for each mutant ( flesh ). Bands were only observed for samples containing 20 ng of the hCA2pET24b DNA template plasmid ( material body ). The size of the bands observed coincides with the size of the plasmid used, 6018 bp.Heat shock transformation and isolation o f plasmid. several(prenominal) colonies were observed after plasmid transformation for two mutants, and 1 colony from each mutant was chosen for restriction enzyme digest with BglII and XhoI.Figure Electrophoretic run on 0.7% agrose gel of DNA of hCAII mutants from PCR mutagenesis reactions. Lane 1 is the GeneRuler ladder by Fermentas and lanes 10-13 are the following V142D (10 ng), V142D (20 ng), H64A (10 ng), and H64A (20 ng). As suggested from the gel, the mutants in the 20 ng plasmid was more than successful than the 10 ng plasmids in determining relative molecular weights. Both mutants in the 20 ng plasmid show a band at approximately the 6000 base pair mark, which coincides with the derive of base pairs in the hCA2pET24b plasmid that was used (6018 base pairs).Quantification of pure plasmid DNA. A 1/twentieth dilution was carried out on the purified DNA with elution buffer (EB 0.1 M Tris, 0.4 M KSCN, pH 7). The absorption of the diluted DNA at 260 nm and 280 nm was taken by a UV/Vis spectrophotometer and the relative DNA purity was determined (Table ). The assumption that Ec1 260 = 50 g/mL for DNA was applied in the calculation of concentrated and diluted concentrations of purified DNA (Table ).Table Relative DNA purity for mutants V142D and H64A determined by UV/Vis spectrophotometer absorbance at 260 and 280 nm. Calculated concentrations of mutants from absorbance data, where Ec1 260 = 50 g/mL.MutantWavelength, Absorbance UnitsRelative DNA Purity (A260/A280)Concentrated (g/mL)Diluted (g/mL)V142D260 nm0.31171.7852311.7015.59280 nm0.1746H64A260 nm0.26531.7581265.3013.27280 nm0.1509Enzyme restriction digest. Purified plasmid DNA of mutants were digested with XhoI and BglII, separately and together in a single and double digest for both mutants. The digested and undigested samples were run on 1% agrose gel, and 2 bands were observed around the 6000 and 7000 bp marker for all 8 samples (Figure , Figure ). The expected length of the bands in the double digest should be 892 bp and 5126 bp (Figure ).Figure Electrophoresis performed in 1% agrose gel of digested V142D hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following V142D plasmid, V142D + XhoI, V142D + BglII + XhoI, and V142D BglII. Double bands are shown at the 6000 and 7000 bp marker for all 4 V142D samples.Figure Electrophoresis performed in 1% agrose gel of digested H64A hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following H64A plasmid, H64A + XhoI, H64A + BglII + XhoI, and H64A BglII. Double bands are shown at the 6000 and75000 bp marker for all 4 H64A samples.Figure Restriction enzyme neglect sites and position of hCAII gene (5072-5854) on the hCAI2pET24b plasmidDNA Sequencing. The mutations for both V142D and H64A in the hCAII gene were successful consort to the sequenced DNA result obtained from ACGT. Other mutations in the DNA sequence were observed in both mutants, th o since the aligned protein sequence was the same, mutations were likely to be silent mutations due to amino acid redundancies. When sequenced in the forward direction by T7 polymerase, a protein mutation was found (K153N) other than the desired mutation of V142D however, when sequenced in the reverse direction by T7 polymerase terminator (T7TER), K153N was not observed.Plasmid DNA transformation into E.Coli BL21(DE3) cells. Following transformation into BL21(DE3) cells, colonies were observed for both hCAII mutants (V142D and H64A). A random colony was chosen to be cultured and then was induced to express protein with 270 M IPTG and 0.1 mM ZnSO4.SDS-PAGE for protein expression. Protein expression was tested with SDS-PAGE. The expected molecular weight of V142D hCAII is approximately 29.2 kDa and the expected molecular weight of H64A hCAII is approximately 29.1 kDa. SDS-PAGE bands are observed between the ladder markers 25.0 kDa and 35.0 kDa for both mutant proteins (Figure , Figure ).Figure SDS-PAGE nasty with V142D hCAII proteins to examine protein expression. Samples were loaded in different volumes of protein to ensure gel visualization. Lane 15 contains the Fermentas protein molecular ladder and lane 1-4 contain the following 1 L IPTG, 4 L IPTG, 1 L+IPTG, 4 L +IPTG. All 4 samples had just about form of protein expression between 25.0 to 35.0 kDa.Figure SDS-PAGE loaded with H64A hCAII protein to examine protein expression. One sample was loaded with 4 L of H64A protein and +IPTG in lane 10. Lane 6 contains the Fermentas protein molecular ladder. The one H64A sample loaded showed an expression between 25.0 and 35.0 kDa.Calculation of pure protein concentration and quenching coefficient. Following affinity purification and dialysis, pure protein concentration was calculated from UV absorption measurements at 280 nm and the known extinction coefficient of hCAII as 50070 M1cm1 (Table ). The final concentration of the samples of V142D and H64A hCAII were 3 .2 M and 37.6 M respectively.Table UV absorption measurements at 280 nm of purified protein and the resulting final concentrationMutantAverage A280Protein concentration (M)V142D0.015833.2H64A0.188437.6SDS-PAGE to assay purity and check approximate molecular weight. Several samples were loaded into the SDS-PAGE for each mutant protein lysate and wash fractions (collected from affinity chromatography), 2 g protein, and 10 g protein. For H64A, a visible band was only observed for the 10 g sample (Figure ). The band was located between the 35 kDa and 25 kDa markers on the ladder. For V142D, none of the 4 samples resulted in a band on the gel (Figure ).Figure SDS-PAGE shown for H64A mutant protein. Lane 1 contains the Fermentas protein molecular weight marker. Lane 11-14 contains H64A samples of the following (in order) lysate, wash fraction, 2 g protein, and 10 g protein. Only the 10 g protein had (faint) observable bands located between the 25 and 35 kDa markers.Figure SDS-PAGE show n for V142D mutant protein. Lane 4 contains the Fermentas protein molecular weight marker. Lane 12-15 contains V142D samples of the following (in order) lysate, wash fraction, 2 g protein, and 10 g protein. No observable bands are seen for any of the samples.Mass spectrometry. ESI-MS was not successful in analyzing the molecular weight of intact and digested protein of both mutants. A MALDI spectrum was able to be generated for the digested proteins however, without the digested ESI spectrum to compare to, the peaks from the MALDI spectrum can only be speculatively assigned.Kinetics Hydrolysis of PNPA. Using the molar absorption coefficient of PNP (1.73-104 M1cm1), the rate of each reaction was determined. The predicted rate was calculated using the Michaelis-Menten kinetics . The plot of predicted rates and actual initial rates vs. PNPA concentration can be seen in Figure , Figure , Figure for wild-type, H64A, and V142D hCAII respectively. The Vmax and KM values for each enzyme wer e calculated by minimizing the square struggle between the predicted and actual reaction rates, and the kcat was calculated using the equation (Table ).Table Calculated Michaelis-Menten parameters for wild-type, H64A, and V142D hCAII catalyzing the hydrolysis of PNPA.Wild-type hCAIIH64A hCAIIV142D hCAIIVmax (M/sec)1.2020.8120.218KM (mM)1.2801.9578.362kcat (s1)5.141 - 1032.159 - 1026.825 - 102kcat/KM (M1s1)4.0211.0328.162Figure Michaelis-Menten plot of initial rate vs. concentration of PNPA added for wild-type hCAII enzyme.Figure Michaelis-Menten plot of initial rate vs. concentration of PNPA added for H64A hCAII enzyme.Figure Michaelis-Menten plot of initial rate vs. concentration of PNPA added for V142D hCAII enzyme.Kinetics CO2 hydration. Initial velocity (V0) values were calculated by measuring rod the progression of the reaction (via concentration of protons) with time (Table , Table , and Table ). kcat values were then calculated using the same equation as in the hydratio n of PNPA and averaged for the individual enzymes (wildtype, H64A, and V142D hCAII) in a particular buffer (i.e. TRIS or imidazole).Table Initial velocity (V0) and kcat values calculated for the hydration of CO2 by wild-type hCAII in TRIS buffer and imidazole buffer.Wild-type concentration (nM)V0 for WT+TRIS (M/s)V0 for WT+Imidazole (M/s)01.3E-086.05778E-081.51.1E-08N/A2.51.1E-085.63E-0852.1E-085.16E-0812.55.9E-085.63E-08Average kcat (s1)5.31.6212.449.19Table Initial velocity (V0) and kcat values calculated for the hydration of CO2 by H64A hCAII in TRIS buffer and imidazole buffer.H64A concentration (nM)V0 for H64A+TRIS (M/s)V0 for H64A+Imidazole (M/s)12.51.4E-086.57E-08251.4E-085.8E-08501.7E-087.53E-08Average kcat (s1)0.670.393.031.97Table Initial velocity (V0) and kcat values calculated for the hydration of CO2 by V142D hCAII in TRIS buffer.V142D concentration (nM)V0 for V142D+TRIS (M/s)12.56.2E-09255.4E-09505.5E-09Average kcat (s1)0.270.19Fluorescence detection of ligand bindi ng. DNSA was titrated with H64A hCAII to determine its affinity for the enzyme. The dissociation constant, KD, for DNSA was determined to be 0.086 M when protein concentration was 0.25 M. Competitive titration of H64A-DNSA hCAII with AZ was attempted, but was not successful as DNSA binding was too tight, making it difficult to be displaced by AZ.Molecular modeling. Literature models of wild-type (PDB code 1CA2) and H64A (PDB code 1MOO) hCAII were analyzed. There is no available structure of V142D hCAII at present. The secondary structure of wild-type is composed of 18 -sheets (77 residues) and 10 -helices (42 residues), with the majority of the -helices travel in the domain of right-handed helices, while very few show left-handed helical properties according to the Ramachadran plot. It also seems that the active site is solely composed of -sheets, and no -helices (Figure ). Analyzing PDB structure 3HS4 (AZ bound hCAII), the mechanism as to how AZ inhibits hCAII function can be see n. AZ has 3 binding sites, 2 are romance binding sites and the other provides a mechanism of inhibition. AZ binds the zinc directly at the active site, displacing crucial ligands needed for catalysis. There were just about discrepancies found between the crystal structure of H64A 1MOO as cited on PDB and nigh mutated H64A from wild-type hCAII, resulting in a RMSD (root mean square deviation) of 0.29 (Figure ). Since no literature structure of V142D is available, no comparison between virtual and crystal structures could be made.Figure Secondary structure of wild-type hCAII overlain with ribbon to visualize the higher arrangement.Figure RMSD between H64A hCAII virtually mutated and literature crystal structure. Blues denote the same or similar residues, while reds and oranges indicate completely different amino acids.DiscussionAgrose gel results were only visible for samples that contained 20 ng of the plasmid template DNA, rather than the 10 ng plasmid. This may be a result of more amplification during PCR with the 20 ng plasmid, and so would intensely be more visible. Though the 20 ng samples showed bands at the appropriate 6000 bp mark, there was also a faint band that can be seen near the end of the gel. This may be due to non-specific primer annealing.Quantification of DNA purity was done by exploiting the peak absorbances of protein and DNA. DNA maximally absorbs at 260 nm, while protein dominantly absorbs at 280 nm. The purity ratio reports the relative amount of DNA compared to protein present in the sample. The purity of both mutants were approximately 1.8, which is regarded as a relatively pure sample however, a purity ratio of more than 2.0 would have been ideal.The restriction enzyme digest showed 2 bands (7000, 6000 bp) for all samples, which may have been a sign of poor mixing/ pipetting since the volumes of restriction enzyme were extremely small amounts. If this is the case, only some of the DNA was nicked and some were not, which would re sult in 2 bands. It was expected that the plasmid sample would have a high band (supercoiled), each of the singly digested samples would have a slightly lower band (nicked), and the doubly digested would show 2 bands that indicated the fragment size of 892 and 5126 bp.Sequencing results showed that a protein mutation occurred when the sample was sequenced in the forward direction by the T7 polymerase. A lysine at position 153 had mutated to glutamine (K153N). However, this mutation was not observed when the T7 polymerase terminator was used to sequence the sample in the reverse direction. A mutation that occurs in one sequencing direction and not the other is usually attributed to sequencing errors, which may be the reason in this case.The SDS-PAGE bands for protein expression coincided with the expected molecular weight for both mutants, which could suggest that the correct proteins were expressed however, there is a possibility that the proteins expressed could be of similar weig ht, but completely different. Interestingly, the V142D samples that did not admit the protein inducer, IPTG, had a more intense band than the faint ones found for the samples that did include IPTG. This may just be a result of mislabelling.The SDS-PAGE performed to assess purity after the purification process. Mutant V142D had low protein expression as evidenced by its concentration of 3.2 M. The V142D mutant should have very low protein expression according to Fierke et al. (1991) because valine at position 142 is uniquely required for maximal expression in E.Coli. It is suggested that by altering position 142, protein stability decreases 2. Therefore, the protein that was expressed in the previous SDS-PAGE gel may not be V142D hCAII at all. The sample may have been small fragmented contaminant proteins that would have completely run off the gel altogether. However, the low concentration of V142D after purification may also be a major factor in the lack of gel bands observed as we ll. Unlike V142D, H64A hCAII concentration should not have affected its lack of bands because it was calculated to have had a reasonable concentration of 37.6 M. There were some problems loading the samples into the wells this could be an explanation as to no observable gel bands.ESI-MS is dependent on concentration because it affects the size of primary droplets 8. The unsuccessful determination of molecular weight of V142D hCAII may be attributed to its low concentration. The H64 hCAII mutant was also not able to be successfully analyzed with ESI-MS. A possible reason for the failure was that it was not kept on ice while it was not being used. The enzyme may have become inactive and degraded into smaller fragments. This would explain the ESI-MS output obtained for H64A. No definite molecular mass was determined, but the spectrometer did detect a lot of small protein fragments in the sample, all under grand piano amu.The kinetic values obtained from PNPA hydrolysis do not follow s imilar trends found in literature 2. The kcat/KM for wild-type hCAII (2500200 M1s1) was found to be significantly big than V142D hCAII (30.3 M1s1) in literature, more than 800- larger 2. Experimental calculations yielded kcat/KM for V142D (8.16 M1s1) to be about 2- larger than wild-type (4.02 M1s1), which did not follow literature patterns. The literature trends make more biolog