Denaturation Of Trypsin Research Articles

Intensive Protein Digestion Using Cross‐Linked Trypsin Aggregates in Proteomics Analysis

AbstractIn this study, cross‐linked enzyme aggregates of tissue‐culture‐grade trypsin (CLEAs‐T) were prepared and introduced to the proteomics analysis instead of the expensive proteomics‐grade trypsin, which benefits from the protein purification during the preparation of the CLEAs. Bovine serum albumin, lysozyme, bovine hemoglobin, and α‐casein were used as model proteins, and three intensive strategies (high‐enzyme‐concentration, high‐temperature, and ultrasound‐assisted digestion) were applied to the rapid and efficient digestion of model proteins by CLEAs‐T. The peptide fragments were then identified by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, which revealed the higher sequence coverage and sharply reduced digestion time of these strategies compared with the conventional in‐solution 12 h digestion method. Morphology studies demonstrated that the excellent performance of CLEAs‐T in proteomics analysis came from the “scaffoldlike” supermolecular structure, which provided a high resistance to the autolysis and denaturation of trypsin under high‐enzyme‐concentration, high‐temperature, and ultrasound‐assisted digestion.

Inertness and stability of a maltose-silica for the separation of proteins by HPLC

A stationary phase consisting of maltose on a silica support, designed for the separation of proteins, is described. When maltose is coupled to aminopropylsilica an effective shielding of the silica surface can be obtained. A size exclusion experiment is used to investigate pore structure and the behaviour of proteins. The stability of the surface layer is studied, as influenced by silica pretreatments and cross-linking reactions. Inertness is studied via the denaturation of trypsin in columns of different length at a number of temperatures. An example of a derivatization of the maltose layer is given which demonstrates its use as an interactive separation medium.

The denaturation of trypsin

The denaturation of α- and β-trypsin in alkaline and neutral solution was studied. The denaturation of α-trypsin was a strict second-order reaction at neutrality. However, the denaturation of β-trypsin was not a pure secondorder reaction at the same pH. Calcium ion retarded the rate of β-trypsin denaturation to a greater extent than that of α-trypsin. In alkaline solution, trypsin has very short half life ( t case1 2 < 30 minutes ). On the other hand, the denaturation of immobilized trypsin in alkaline solution is a first-order reaction as is immobilized chymotrypsin. The rates of these two denaturations are similar. Calcium ion does not affect the rate of trypsin denaturation in alkaline solution.

トリプシンの尿素変性に対する界面活性剤の影響(第1報)

尿素によるトリプシンの変性失活に対するsodium dodecyl sulfate (SDS)の影響を, SDSのトリプシンに対する比率を変え,中性付近で検討し次のような結果を得た. (1) 尿素が低濃度(3M)のときはSDSはトリプシンに対し,尿素のない場合とほとんど同様の変性作用を示すが,高濃魔(9M)尿素の存在下でSDS/トリプシン比が2～4の範囲では,トリプシンの尿素による不可逆的失活を阻止する,上記比率以下では著しい影響はみられず,以上ではSDSによる失活が一応優先する. (2) トリプシンのSDSのみによる失活は,中性付近ではSDS/トリプシン比がやはり2～4の間で最大である.したがってSDSがトリプシンを失活させる場合のSDS/トリプシン比と,トリプシンの尿素による不可逆的失活を阻止する場合の比はほぼ等しい. (3) 結局上記の条件下で9M尿素と,トリプシンに対し2～4倍の濃度で含まれるSDSとは,互いに他のトリプシン変性作用を阻止することが認められた.