Introduction
Initially there were beliefs that, MMPs are jointly able to degenerate all constituents making up the extracellular matrix (ECM) together with basement membrane that confines their operations to tissue reconstruction as well as the preservation. Nonetheless, the contemporary studies also signify that MMP-1 is responsible for controlling the production or else the activation of the chemokines. At this point, it is critical to understand that MMP-1 is among other MMPs.
Similarly, the knowledge holds it that MMP- 1 cleaves a peptide bond beforehand a residue comprising of a hydrophobic adjacent chain, for instance Met and Leu. The other sideways chains cleaved include Ile, Tyr as well as Phe. There is a seldom cleave on a peptide bond containing a charged residue at this point, with the cleavage of the X-Lys bond performed by MMP -1. The hydrophobic remains appropriate into the S1 specificity pocket that has their size and shapes varying substantially amongst MMP- 1. Moreover to the S1_ pocket, other substrate contact locations and subsites correspondingly engage in the enzyme’s substrate precision. In given situations, noncatalytic domains affect the enzyme activity, especially contrary to large extended macromolecules of the ECM. For instance, the fibronectin domains of MMP-2 together with MMP-9 are important for its activity on type IV collagen, gelatin, and elastin.74,75 In collagenase 1 (MMP-1), the loop section adjacent to the catalytic site helix is vital for collagenolytic operation. Additionally, the hemopexin domain together with the hinge amid the catalytic with the hemopexin domains also performs vital functions in collagenolysis.
MMP-1 acts as regulators of the extracellular tissue signalling pathways. As a result of the wide network emanating from their substrate specificity, the MMP-1 promote the homeostasis of several tissues besides participating in numerous physiological operations, for instance bone modification, immunity, wound remedial of wounds and angiogenesis. MMP-1 operations are strictly regulated at the transcription point, pro-peptide activation as well as suppression by tissue inhibitors of the MMPs. Improper control of the MMP-1 activity results into pathological scenarios for instance, arthritis, inflammation in addition to cancer. This also poses MMP-1 as favourable therapeutic marks.
Even though the operations of MMP-1 among other MMPs have been observed to be crucial in cell biological activities together with essential physiological occurrences incorporating tissue modification, such as bone modification, immunity, wound remedial of wounds and angiogenesis they also associate many pathological conditions involving inflammatory infections and cancer.
Objective
The purpose of this experiment is to establish the usage of nickel (Ni2+) affinity chromatography by using it to distinct two domains created from the breakdown of a medically-essential protease that is matrix metalloproteinase-1 (MMP-1) in the preparative separation of proteins. The exercise also intended to determine the domain that contains the Ni2+-binding activity between the two in MMP-1.
The affinity chromatography incorporates a technique for separating biochemical substances depending on a highly specific association for instance the interaction between an enzyme together with a substrate. This system associates two phases, the mobile and the stationary phase. The immobile phase comprises of a gel matrix. The initial point involves indeterminate heterozygous compound of molecules in the solution.
The hydrolytic activity of the CAT domain executed in the thiopeptide enzyme assay follows a number of mechanisms for quantitation in MMP- 1. The thiopeptide substrate contains a sulphur atom which replaces the amide group of the scissile bond. In case hydrolysed, the C-terminal portion of the thiopeptide substrate contains a free thiol group (-SH). The content of free thiol groups (and thus the activity of the enzyme) can be quantitated through its reaction with 5, 5’- dithiobis[2-nitrobenzoic acid] (DTNB), normally referred to as Elman’s reagent. In this reaction yellow product that can be quantified through spectrophotometry.
Experimental Procedures
Reagents.
The thiopeptide substrate Ac-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gly-OC2H5 was supplied by ENZO Life Sciences. All other biochemicals were bought from Fisher Scientific. Recombinant MMP-1 was produced as described previously (Arnold et al., 2011). Autolysis of the integral enzyme into its composite catalytic (CAT-1) and hemopexin (HPX-1) domains was carried out through incubation of the enzyme for 24 hours at 37°C in sample buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10 mM CaCl2).
Affinity Chromatography
The CAT-1 and HPX-1 domains were separated by exploiting the natural affinity of MMP-1 for immobilised Ni2+ ions. Following autolysis, the mixture was applied to a 1 mL HiTrap IMAC FF column that had been altered in prior using 0.1 M NiSO4. The non-binding protein was splashed through the column with 5 mL sample buffer. The bound protein was eluted with 5 mL elution buffer (sample buffer supplemented with 500 mM imidazole). In each case, 1 mL fractions were collected for quantification of protein concentration using the Bradford assay. From each chromatography step, fractions found to contain more than 50 µg/mL protein were pooled into either the non-bound fraction (NBF) or eluted bound fraction (EBF) for subsequent quantification of proteolytic activity.
Bradford Assay for Protein Quantification
A 100 µL aliquot of each sample was mixed with 900 µL of Bradford reagent (0.1 mg/mL Coomassie, 5% MeOH, 8.3% H3PO4), incubated for 5 min, and then the A595 was measured in a spectrophotometer. Protein concentrations were determined from these values by reference to a standard curve of 0 to 200 µg/mL bovine serum albumin (BSA).
Thiopeptide Proteolysis Assay
The level of proteolytic activity in the original mixture, NBF and EBF was measured from hydrolysis of the thiopeptide substrate in assay buffer (50 mM HEPES pH 7.4, 10 mM CaCl2, 0.05% (w/v) Brij-35, 1 mM 5,5’-dithiobis[2-nitrobenzoic acid] (DTNB)). Each thiol-containing C-terminal product molecule reacts with DTNB to produce an NBS- ion which absorbs at 412 nm. Hence, substrate hydrolysis was quantitated using a spectrophotometer by the increase in A340 per min (ΔA340 / min) throughout the first minute of the reaction. The outcome was then changed into the units of enzyme (µmol substrate consumed / min / mL) through the molar extinction coefficient for the NBS- ion that is, ε412 = 13,600 cm-1 M-1.
Results and Conclusions
The bovine serum albumin reacts in the solution to yield a complex. The observations made are based on the intensity of the complex as it is read in the spectrophotometer. The quantities observed are proportional to the yield made in the entire solution. There are variables in this experiment and the two main determinants are the fractional yield as well as the time span during the experiment.
Starting with the Non Bound Fraction, the elution rate is constant. There is a constant trend manifested in the reaction. The duration is determined in contemplation with the production of the complex that might be eluted or not eluted either. The time is in predetermined number of seconds. The preferable time is between five and ten seconds. The span is to be a significant period to allow for the measurement of critical amount of the substance produced in the solution. The NBF attains a higher yield of 0.162 mL at the end of the 18th measurement exercise. There is a higher rate of reaction between the enzyme and the substrate.
Consider the Eluted Bound Fraction that is also a subject to reaction with the substrate. Within the first few seconds probably three, the reaction yields an amount of 0.002 mL of the product. At the end of the last measure of the produced material, there is 0.009mL produced. The amount produced is smaller, however the entire yield depends on the reaction in the mixture and the formation of the complex. In the category of the enzyme reaction there is the reaction of mixture with the substrate. The thiopeptide exposed to enzyme activity produces the complex at a dissimilar rates of the bound and non- bound fractions. The first reaction produces a yield of 0.020 mL and the last measurement depicts a yield of 0.157 mL.
Since there were three different reactions of compounds with one solvent, a comparison can be made and the special property amongst the m can be determined. Consider drawing graphs of the three sets of results against time that can act as a non- variant component. As it can be seen in the graphs there is consistency of the results as they are read. The three graphs manifest gradients that can in turn be termed as the coefficient of the proteins in their reactivity. The Non Bound Fraction of the MMP 1 macromolecule is highly reactive. The coefficient obtained in the graphs are more practical as they can be used to prove theories postulated by different scholarly postulations.
The calculations made show a coefficient of one that is uniform in all the reactions and the curve is a straight line. Basically, the reactions are constant amongst the three solutions. However, since there are different constituents the output of the targeted substance for measure is different. Different samples have different concentrations and this translates into dissimilar fractions of observable fractions of A in the reactions.
Separation of MMP-1 Domains by Ni2+-Affinity Chromatography
The MMP-1 contains two main constituent namely the catalytic (CAT-1) and hemopexin (HPX-1) domains. The Ni2+-Affinity Chromatography endeavours to analyze the MMP-1 through its constituents for t6the stationary Ni2+ ions. The ions determine the mobile and the static phases that then define the elution of the constituents. The autolysis targeted in this phase of the experiment concerns the breaking down of the mixture by the enzyme itself. Therefore, a separation takes place though the phase of MMP – 1.
There were two observable peaks as the outcome in the experiment. The first peak is at fraction number 2 and at a maximum protein concentration of 92. The second peak is at fraction number 8 and with a maximum protein concentration of 224.
The fractions pooled to make the NBF were fraction 8 whilst the fraction 2 was pooled to make EBF.
The NBF fraction contained a significant amount of Enzyme activity as it is observable in the results. The NBF has a steeper gradient of reaction as compared to the EBF that signifies a less steep gradient and low rates of activity among its constituent reactants. Consider the reaction results of the two bound fractions.
Non Bound Fraction corresponds to catalytic (CAT-1) domain whilst the Eluted Bound Fraction corresponds to the hemopexin (HPX-1) domain.
Original = 1.288
NBF = 283 0.291
EBF = 889 0.516
Quantification of CAT-1 Domain Purification
Graph of E against Time
Calculating the coefficient of E
Coefficient = Change in EChange in Time
= 0.18 –0.0618 - 6
= 0.1212
= 0.01
Graph of N against Time
Calculating the coefficient of N
Coefficient = Change in NChange in Time
= 0.18 –0.0618 - 6
= 0.1212
= 0.01
Graph of Original Mixture against Time
Coefficient = Change in Original mixtureChange in Time
= 0.18 –0.0618 - 6
= 0.1212
= 0.01
Non Bound Fraction (NBF)
Activity = (ΔA412/min x pathlength / ε412) x (reaction volume) / (enzyme volume)
The ΔA412/min = (0.162 / 3 min ) = 0.034 min-1
NBF, the ΔA412/min = 0.034 min-1, then:-
Activity = (0.034 min-1 x 1 cm / 13,600 M-1 cm-1) x 0.001 L / 0.005 mL
= (2.5 x 10-6 M/min) x 0.001 L / 0.005 mL
= (2.5 x 10-9 mol/min) / 0.005 mL
= (0.0025 μmol/min) / 0.005 mL
= 0.0025 U / 0.005 mL
= 0.5 U/mL
Activity Yield = new activityinitial activity
= 0.5 U/mL0.5 U/mL
= 1
Protein
= total protein (mg)/volume (vL)
= 0.291 mg/ 1.5 mL
= 0.859 mg/mL
Specific activity
= Total activity (U)/ Total protein (mg)
= 2.0 / 0.291
= 6.87
Protein Activity
= total activity (U)/ volume (mL)
= 0.5 U / 0.5 mL
= 1UmL-1
For EBF
The ΔA412/min = (0.010 / 3min ) = 0.003333 min-1
EBF, the ΔA412/min = 0.003333 min-1, then:-
Activity = (0.003333 min-1 x 1 cm / 13,600 M-1 cm-1) x 0.001 L / 0.005 mL
= (2.4 x 10-7 M/min) x 0.001 L / 0.005 mL
= (2.4 x 10-9 mol/min) / 0.005 mL
= (0.0024 μmol/min) / 0.005 mL
= 0.0024 U / 0.005 mL
= 0.48 U/mL
Activity Yield = new activityinitial activity
= 0.48 U/mL0.48 U/mL
= 1
Protein
= total protein (mg)/volume (vL)
= 0.516 mg/ 0.5 vL
= 1.08 mg/vL
Specific activity
= Total activity (U)/ Total protein (mg)
=2.0 / 0.516
= 0.48 U/mg
Protein Activity
= total activity (U)/ volume (mL)
= 2.0 / 0.5
= 0.2 U/mL
Main
Activity = (ΔA412/min x pathlength / ε412) x (reaction volume) / (enzyme volume)
The ΔA412/min = (0.157 / 3min ) = 0.052 min-1
Main, the ΔA412/min = 0.052 min-1, then:-
Activity = (0.052 min-1 x 1 cm / 13,600 M-1 cm-1) x 0.001 L / 0.005 mL
= (3.2353 x 10-6 M/min) x 0.001 L / 0.005 mL
= (3.2353 x 10-9 mol/min) / 0.005 mL
= (0.0032353 μmol/min) / 0.005 mL
= 0.00323 U / 0.005 mL
= 0.64 U/mL
Activity Yield = new activityinitial activity
= 0.64 U/mL0.64 U/mL
= 1
Protein
= total protein (mg)/volume (vL)
= 1.288 mg/ 1.5 vL
= 0.96 mg/vL
Specific activity
= Total activity (U)/ Total protein (mg)
= 0.96 / 1.288
= 0.745
Protein Activity
= total activity (U)/ volume (mL)
= 0.00323 U / 0.005 mL
= 0.64 U/mL
Discussion
Increase in A595 fraction with increase in BSA
The A595 fraction increases with the rise in the BSA. In the making of standard, the BSA solution availed was diluted with 200 μL of that solution with 800 μL of loading/wash buffer. The A595 fraction is produced by the reaction between the BSA and the Bradford reagent as well as the samples. The fraction is determined using a spectrophotometer with a blank of the BSA standard.
The Bradford Reagent is fundamental in determining the concentration of proteins in various mixtures or solutions. The process relies on the development of a complex between the dye, and proteins in the analysed solution. The protein-dye complex results into a shift in the absorption maximum of the dye probably from 465 to 595 nm of the spectrophotometer. The quantity of absorption is relative to the protein available in the solution. The Bradford Reagent needs no dilution and is appropriate for the various forms of processes and assays such as micro, multi-well plate, together with standard assays. Similarly, the Bradford Reagent is companionable with the reducing agents that are normally applied in stabilizing proteins in the mixture. In this case, the Bradford Reagent was appropriate because it is only compatible with low concentrations of detergents and that was the scenario. The spectrophotometer operates on the principle of absorption. Therefore, the proportionate increase in the observable fraction was due since it tantamount to the increase in the intensity of the dye with the protein forming the complex in the reaction.
A yellow product observed
During the reaction, a yellow component was seen. This resulted from the reaction of the substrate with Elman’s reagent (5, 5’-dithiobis [2-nitrobenzoic acid] (DTNB)). The thiopeptide has a C-terminal fragment which contains which has ahas a sulphur atom replacing the amide group of the scissile bond. MMP 1 is multi-domain enzyme which is made up of a pro domain that is, CAT - 1 domain, a linker portion plus a HPX-like domain. The cleavage of the pro domain results into a remarkable rearrangement of the N-terminal residues (Arnold et al, 2011). The activation process swings Phe81 towards the proteinase domain and terminates with a salt link between the amine group and the carboxylate side-chain (Arnold et al, 2011). This connection conveys numerous-fold higherr enzymatic activity to the MMP-1 as compared to those possessing either an extended or a shorter N terminus that has no salt linkage (Arnold et al, 2011). Formerly an inhibitor-free structure of the catalytic domain of MMP-1 was reported (Visse & Nagase, 2003). This conformation was, nevertheless, distinct to the extent that the N-terminal Leu-Thr-Glu-Gly (Visse & Nagase, 2003) components of one fragment engaged the active portion of the other molecule hence forming an unusual inhibited complex that has not categorically be well-thought-out as an inhibitor-free structure (Visse & Nagase, 2003). The current orientation alternatively, poses unique in being elucidated without an inhibitor. This is the observable structure of the full-length MMP-1 without ligand and it is the scenario in which a water molecule is observed at the active position availing the 4th ligand for the catalytic zinc in the tetrahedral organization sphere (Visse & Nagase, 2003).
The Ni2+ binding site on MMP – 1
The Ni2+ binding site on MMP – 1 is on the zinc motif. The catalytic domain is an oblate sphere with the cross-sectional groove as its active site for binding. On this active site, there are Zn2+ ions that are bound by histidine components found in the reserved section of HExxHxxGxxH reserved sequence (Fig 2). It is at this point where it was found.
The Specific Activity, Purity and Yield
The specific activity incorporates the enzyme’s activity in each milligram of the total protein. The affinity chromatography produces critical specific activity with reference to the entire protein. The activity is higher in the total protein in the solution. The specific activity in the method is significant due to the higher total activity. The fold purity obtained using the method is beyond the threshold and can be substantiated in the report since the domains eluted have insignificant residues of impure components and are assumed to be nonexistence. The yield ranges from >90% and in this case it was 96% to 100 and more. Therefore, affinity chromatography is a critical technique of determining the components of different domains of the enzyme.
The Automated, Quantitative Protein Purity Analysis with SDS-Gel Capillary Electrophoresis can be used as well in assessing the level of purity of the CAT – 1 and HPX domains since it allows for the coexistence of proteins with different conformations.
Improving the Results
The improvement of the results can be attained through a number of steps such as (i) ensuring low rates of errors especially during reading of the quantities, (ii) using properly calibrated instruments and (iii) using appropriate procedures during the experiments together with accurate computation of the results.
Figure 1 The crystal structure of MMP-1. Insert a diagram showing the three-dimensional structure of MMP-1, highlighting the domain structure and the catalytic cleft. Ensure that it is annotated appropriately, and that you cite the correct reference for the image (unless you have produced it yourself).
Figure 2. The thiopeptide assay used to detect MMP-1 CAT domain activity. Insert a diagram showing the thiopeptide substrate, the cleavage products produced by MMP-1, and the subsequent reaction with DTNB. Finally, complete this figure legend appropri
Supplementary Information
The BSA standard Curve
Examples of calculations for enzyme activity, specific activity and fold purity
Non Bound Fraction (NBF)
Activity = (ΔA412/min x pathlength / ε412) x (reaction volume) / (enzyme volume)
The ΔA412/min = (0.162 / 3 min ) = 0.034 min-1
NBF, the ΔA412/min = 0.034 min-1, then:-
Activity = (0.034 min-1 x 1 cm / 13,600 M-1 cm-1) x 0.001 L / 0.005 mL
= (2.5 x 10-6 M/min) x 0.001 L / 0.005 mL
= (2.5 x 10-9 mol/min) / 0.005 mL
= (0.0025 μmol/min) / 0.005 mL
= 0.0025 U / 0.005 mL
= 0.5 U/mL
Activity Yield = new activityinitial activity
= 0.5 U/mL0.5 U/mL
= 1
Protein
= total protein (mg)/volume (vL)
= 0.291 mg/ 1.5 mL
= 0.859 mg/mL
Specific activity
= Total activity (U)/ Total protein (mg)
= 2.0 / 0.291
= 6.87
Protein Activity
= total activity (U)/ volume (mL)
= 0.5 U / 0.5 mL
= 1UmL-1
For EBF
The ΔA412/min = (0.010 / 3min ) = 0.003333 min-1
EBF, the ΔA412/min = 0.003333 min-1, then:-
Activity = (0.003333 min-1 x 1 cm / 13,600 M-1 cm-1) x 0.001 L / 0.005 mL
= (2.4 x 10-7 M/min) x 0.001 L / 0.005 mL
= (2.4 x 10-9 mol/min) / 0.005 mL
= (0.0024 μmol/min) / 0.005 mL
= 0.0024 U / 0.005 mL
= 0.48 U/mL
Activity Yield = new activityinitial activity
= 0.48 U/mL0.48 U/mL
= 1
Protein
= total protein (mg)/volume (vL)
= 0.516 mg/ 0.5 vL
= 1.08 mg/vL
Specific activity
= Total activity (U)/ Total protein (mg)
=2.0 / 0.516
= 0.48 U/mg
Protein Activity
= total activity (U)/ volume (mL)
= 2.0 / 0.5
= 0.2 U/mL
Main
Activity = (ΔA412/min x pathlength / ε412) x (reaction volume) / (enzyme volume)
The ΔA412/min = (0.157 / 3min ) = 0.052 min-1
Main, the ΔA412/min = 0.052 min-1, then:-
Activity = (0.052 min-1 x 1 cm / 13,600 M-1 cm-1) x 0.001 L / 0.005 mL
= (3.2353 x 10-6 M/min) x 0.001 L / 0.005 mL
= (3.2353 x 10-9 mol/min) / 0.005 mL
= (0.0032353 μmol/min) / 0.005 mL
= 0.00323 U / 0.005 mL
= 0.64 U/mL
Activity Yield = new activityinitial activity
= 0.64 U/mL0.64 U/mL
= 1
Protein
= total protein (mg)/volume (vL)
= 1.288 mg/ 1.5 vL
= 0.96 mg/vL
Specific activity
= Total activity (U)/ Total protein (mg)
= 0.96 / 1.288
= 0.745
Protein Activity
= total activity (U)/ volume (mL)
= 0.00323 U / 0.005 mL
= 0.64 U/mL
References
Arnold et al (2011) describes the production of MMP-1 in the Pickford lab, Iyer et al (2006) describes the crystal structure of MMP-1, and the rest are reviews of MMPs.
Arnold LH, Butt LE, Prior SP, Read CM, Fields GB & Pickford AR (2011). The interface between catalytic and hemopexin domains in matrix metalloproteinase-1 conceals a collagen binding exosite. J. Biol. Chem. 286, 45073-45082.
Brinckerhoff CE & Matrisian LM (2002). Matrix metalloproteinases: a tail of a frog that became a prince. Nat. Rev. Mol. Cell Biol. 3, 207-214.
Iyer S, Visse R, Nagase H & Acharya KR (2006). Crystal Structure of an Active Form of Human MMP-1. J. Mol. Biol. 362, 78-88.
Nagase H, Visse R & Murphy G (2006). Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc. Res. 69, 562-573.
Visse R & Nagase H (2003). Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Cardiovasc. Res. 92, 827-839.
