Isolation,Purification And Interactions Of Myofibrillar Proteins Extracted From Jumbo Squid (Dosidicus Gigas)

As giant squid(Dosidicus gigas)is abound with low price and high nutritional value.Thus it is an promised candidate serving as a raw mate-rial surimi and other meat products,but with remarkable shortcoming of poor gel-forming capacity.As a result,it is still unpopular in surimi in-dustry.So it is urgent to improve the gel performance of giant squid myo-fibrillar protein in order to enhance the quality of surimi product.Since protein is macromolecule with complicated structure and diverse func-tions.It is well-known that the protein structure modification always leads to its properties change,which will affect the product quality.Therefore,it is the start point to analyze the relationship between protein structure and function in this thesis.Pure proteins have been isolated and purified from complex myofibril protein system.Then these pure proteins have been served as a model to study of the relationship between protein strcture and gel properties.And these pure proteins mixed together to mimic myofibrillar protein and its phycial and chemical properties have been studied in order to purchase the protein mixture system.This paper focused on the establishment of methods for the isolation and purification of high purity single proteins from giant squid myofi-brillar proteins.The myofibrillar proteins were first separated by HiTrap Q FF 16/10 column and then were done further by SuperdexTM 20010/300 gel column with the AKTA purifier system.With these procedures,four high purity proteins:myosin,actin,tropomyosin and myosin light chains,have been obtained,which were attested by MALDI-TOF-MS,SDS-PAGE and HPLC with the result of paramyosin 85%,actin 92%,tropomyosin 98%and myosin light chain 96%.During the process of protein purification,it was found that when protein solutions collected from the ion column by ultrafiltration concen-trated,there are a lot of fiber filamentous aggregates in protein solution.Here the protein solutions of the flowthrough peak A and the 30%elution peak D were collected,and the fiber filamentous aggregates are very reg-ular and homogeneous.Given the formation of aggregates may have an impact on the separation and purification of proteins,the formation mechanism of the aggregates was probed in this thesis.Firstly,the micro-structure and particle size of the protein were characterized by fluores-cence microscopy and dynamic light scattering respectively.The results showed that the formation of the fiber aggregates was formed after ultra-filtration.The conformational change of protein before and after ultrafil-tration concentration was studied at the molecular level by UV,fluores-cence and circular dichroism techniques.The results showed that protein molecules formed a regular fibrous structure with large particle size,and the spatial conformational change occurred after ultrafiltration concentra-tion.More tryptophan and tyrosine residues were exposed to solvent,and the content of α-helix secondary structure increased.As a result,the pro-tein structure becomes more orderly.It is suggested that the formation mechanism of fiber aggregates is mainly driven by the concentration ef-fect and the shear effect.Since with protein concentrated,the probability of protein molecules colliding increases.This perfects protein aggrega-tion.The shear effect may lead to the change of protein conformation,which increases the hydrophobic interaction and hydrogen bonding be-tween proteins.Protein particles form a rod-like structure in a specific linear connection due to the fact that each short rod may contain only two specific linking sites at each protein molecules.It is presumed that fibril aggregates are formed by paramyosin and actin.As the gel formation process is closely related to the interaction be-tween protein molecules and the gel properties are related to salt concen-tration,so it’s essential to study the interactions of protein molecules.In this thesis,the purified paramyosin as the model was explored for this is-sue.The particle size and weight-average molecular weight of the purified protein were characterized by light scattering technique.The results showed that the weight average molecular weight of paramyosin was 207.41 ± 20.14 kDa,the average hydraulic radius was 18.65 nm,and the second virial coefficient A2 of paramyosin was negative,and decreased with the increase of KCl concentration,which indicates that the interac-tion forces between the paramyosin molecules is dominated by the dou-ble-layer force.With the increase of KCI concentration,this force reduces,and the protein tends to aggregate.