Proteolysis of the protein hydrogel formed by PCLP and CCLP by collagenases will cause exposure of the protomers and drug release at the site. This important aspect of this responsive material requires sensitivity to relevant proteases, and is the specific subject of the current study. MMPs also aid in the anti-inflammatory response, when the stimulation of the inflammatory response has subsided, by removing inflammatory cells from the healed wound5.
In addition, the inflammatory response increases the acidity of the surrounding ECM, which also plays a role in the pathological response8. Studies have shown that decreased pH, which is a product of the inflammatory response, also has an effect on pathological aspects of inflammation. There are two stages of the inflammatory response, depending on the length of time that pathological effects persist.
The therapeutic benefit comes mainly from the ability of NSAIDs to block specific prostaglandin synthesis, through inhibition of the. cyclooxygenase enzymes COX-1 and COX-2. In order to effectively circumvent the systemic side effects that occur when NSAIDs are administered orally, specific conditions of the site of inflammation can be exploited to deliver drugs locally. Changing drugs so that excretion is possible is mainly the work of the liver and kidneys.
After the intended effects of NSAIDs, the kidneys must also work to clear them from the blood. In solution, the central helix adopts an unstructured conformation16, which allows Calmodulin-Ca2+ complexes the freedom to fully conform to the structure of the target peptide (Figure 1.2). Schematic depiction of the Calmodulin-Ca2+ complex with the M13 peptide depicts the conformation of the Calmodulin-Ca2+ domain tightly with the M13 domain.
Finally, initial studies of the proteolytic degradation of the gels themselves as a function of pH and time. After decanting the supernatant, we added 15 ml of 10% Triton-X to the pellet, stirred the pellet, and incubated it for 10 min at room temperature. Initially, we planned to use MALDI-TOF to identify the precise molecular weights of each respective band, but the negative mode of the MALDI-TOF machine was not.
As seen in graphs 2.1 and 2.2, we created a trend line from the standard protein scale by plotting the logarithmic base 10 of the molecular weight of each protein band vs. Using the equation of the trend line, we were able to accurately identify the molecular . Figures 2.2, 2.3 and 2.4 show the results of the pH dependence of CCLP3 proteolysis by Collagenase.
The base 10 logarithm of molecular weight plotted against the relative mobility of the standard MW protein ladder to produce a trend line.
CCLP3 Collagenase Digest
The equation for the trend line is then used to determine the molecular weight identities for the major bands. As such, proteolysis of this construct creates a feathered appearance for the low MW fragments and no high MW fragments as observed in the CCLP3 digest. Calmodulin sequences cleave CCLP2 in half and then there is cleavage of other CLS sites, creating feathery and higher MW intermediates as the CLS sites are cleaved.
Catalytic efficiency at pH 6.5 is difficult to observe due to cleavage of indistinguishable CCLP2 backbone-forming band fragments. The trend line formed from the gel migration patterns on the protein ladder for examining CCLP2 fragment identification.
CCLP2 Collagenase Digest
CONCLUSION
SDS-PAGE of collagenase digestion of CCLP3 reveals that the enzyme showed minimal activity at pH 5.7, while proteolysis experiments performed at pH 6.5 gave optimal results, cleaving the indicated site in the shortest time, with proteolysis at pH 7.4 as a control. It is difficult to make absolute statements regarding the pH dependence of CCLP2 due to fragments of the main band of CCLP2 disappearing in both guts after about 80. To optimize stepwise, pH-dependent drug delivery, the CCLP3 construct is probably the best of our current delivery system constructs hydrogel-based drugs.
To accurately characterize fragments produced from the cleavage of collagenase, trend lines were formed from the known molecular weight ladder. With the trend line and known molecular weights of expected cleaved constructs, exact identities of bands were determined. The 16,900 D band can be distinguished as Calmodulin, and the band corresponding to 20,300 D is Calmodulin with the leader sequence manipulated in the protein for His-Tag purification.
Due to the many different cleavage points present in CCLP2, there are several different bands that can form with similar molecular weights. Furthermore, because the single-band resolution of the digested construct is poor, it is not possible to conclude which exact calmodulin-incorporating construct was formed. During CCLP2 SDS-PAGE characterization, final 1280-min samples were treated with EDTA to chelate Ca2+ from.
By observation, we can conclude that Ca2+ did not alter the migration of the fragments to any appreciable amount. We started investigating pH-dependent matrix metalloproteinase-specific catalysis of an engineered protein substrate, but the project was halted due to gel anomalies. Activation of MMP-1 occurs by cleavage of the 52-57 kD propeptide form, with the molecular mass of MMP-1 changing by approximately 10 kD, resulting in the active form of 42-47 kD, depending on glycosylation.
Activation of MMP-8 occurs through cleavage of the 85 kD inactive form to produce an active 65-70 kD activated form. Despite a noticeable difference in molecular weights between the active and inactive forms of each respective MMP, it is evident from Figure 2.7 and Figure 2.8, SDS-PAGE of the CCLP3 proteolysis experiments, that each band, whether inactive or active, and whether MMP-1 or MMP-8, are strikingly similar in molecular weight. This warrants further research into the activation of MMP-1 and MMP-8, as well as the activity on physiological.
