CFR11-Protease Lab Report

The pH is another important factor that influences the enzyme activity, as it affects the shape of the enzyme or charge on substrate and ultimately its functionality (Reece et al., 2011; Eed and John, 2013). With respect to effect of pH on enzyme activity, there was a steady increase in the activity for CFR15-protease, i.e. from pH 3.5 to 10.5 and a sudden fall was observed over pH 10.5. The activity was stable in the range of pH 7 to 10.5 and this reveals the alkaline protease nature of the enzyme. However, CFR11-protease showed increase in the activity from the pH 3.5 to 5.5 and nearly stable activity in the range of pH 5.5 to 7.5. There was a steep fall in the activity over pH 7.5 and the maximum activity was observed at pH 7.4. These observations
At 24th hour incubation, the degradation product with the size of 20.1 kDa appeared and no further degradation was observed. Among four chains of fibrin, only α-chain was degraded completely and other three chains (α-polymer, γ-γ’ dimer and β-chains) remained intact. Similar to that of CFR15-protease, CFR11-protease also produced few degradation products (n=2) with approximate size in the range of 20.1 kDa. This clearly implied the endopeptidase nature of the enzyme. The degradation pattern of fibrin by CFR11-protease evidently indicated that only α-chain was specifically and preferentially degraded by the enzyme over the other chains of fibrin. This represents the specificity of the enzyme. This observation is significant significant, since the insoluble fibrin formation is the result of cross linking seen in α-chains during polymerization (McKee et al., 1970). Hence, search for the fibrinolytic protease with precise action to α-chain is most vital.
The multiple proteases of BR21 could degrade all the four chains of fibrin effectively and rapidly. There were more number of low molecular weight degradation products accumulated below the β-chain and rapid degradation of all the chains of fibrin was seen within 3 h of incubation. The size of the degradation products ranged from 29 to 14.3 kDa. Except for CFR11-protease which
A common observation in this regard is that, Aα and Bβ chains of fibrinogen were degraded rapidly where as, γ-chain of fibrinogen showed significant resistance to degradation in all the three cases of CFR15-protease, CFR11-protease and multiple proteases of BR21 as evident from SDS-PAGE analysis of fibrinogen degradation. Higher concentration of fibrinogen and glycosylation of fibrinogen are the major risk factors in conditions of atherosclerosis, thrombosis and diabetes (Lassila et al., 1993; Smith et al., 1986). Hence, property of fibringenolytic activity is an additional benificial attribute apart from fibrinolytic activity. This nature of the enzyme may help in thining of blood by the degradation of fibrinogen and may reduce the risk of thrombosis which is due to formation of undesirable fibrin. MS/MS anlaysis or tandem mass spectroscopy has profoundly influenced the field of protein science. Being helpful in understanding the sequence of peptides for identification of proteins, it has been employed in many areas of proteomics (Mann e t al., 2001; Chandrasekaran and Sathyabama,