Denatured protein solubilized solution

Abstract

To provide a denatured protein solubilized solution capable of solubilizing thermally-denatured protein.SOLUTION: A denatured protein solubilization solution contains surfactants, reducing agents, and water. As surfactant components, it contains either sodium dodecyl sulfate at a blending concentration of 1% to less than 10%, Triton X at a concentration of 1% or more and 10% or less, or Tween 80 at a concentration of 1% or more and 10% or less. As reducing agent components, it contains either 1-thioglycerol at a concentration of 0.1% or more and less than 10%, thiourea at a concentration of 2.5% or more to 5% or less, dithiothreitol at a concentration of 0.1% or more and less than 10%, or sodium 2-mercaptoethanesulfonate at a concentration of 0.5% or more and less than 10%, and has a pH of 4 or more and 8 or less.

Description

[Technical field]

[0001] The present invention relates to a denatured protein solubilizing solution capable of solubilizing heat-denatured proteins. [Background technology]

[0002] Since surgical instruments used in surgery have a large amount of blood and body fluids attached to them, it is very important in terms of infection control to thoroughly clean and sterilize them. In addition, cleaning evaluation must be performed to evaluate the cleanliness of the instruments after cleaning. Cleaning evaluation is an evaluation of the cleanliness of the instruments based on the amount of residual protein, using the protein in blood remaining on the instruments as an indicator. Non-Patent Document 1 has a section on cleaning evaluation and describes the detailed method.

[0003] Incidentally, when performing cleaning evaluation, a surfactant, sodium dodecyl sulfate (hereinafter, SDS), is used to extract proteins remaining on the equipment, but it is a well-known fact that SDS cannot extract denatured proteins. Therefore, while cleaning is often performed at a high temperature of about 90°C in the actual cleaning process, it is described that cleaning evaluation requires cleaning at a temperature lower than 60°C, at which proteins do not denature (see Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Japan Society of Medical Devices, Guidelines for Sterilization Assurance in Medical Settings 2021 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, when conducting cleaning evaluation, it is necessary to conduct the evaluation in a different cleaning process from the actual one, and there are doubts as to whether the desired cleaning process is truly evaluated correctly. Also, some types of cleaning machines are only compatible with high-temperature cleaning, and the current situation is that cleaning evaluation cannot be performed.

[0006] Therefore, the inventors believed that in order to properly evaluate the cleaning of equipment cleaned in an actual cleaning process, a solution capable of extracting thermally denatured proteins was necessary, and thus developed the present invention.

[0007] That is, an object of the present invention is to provide a denatured protein solubilizing solution capable of solubilizing heat-denatured proteins. [Means for solving the problem]

[0008] The above object of the present invention can be achieved by the following means.

[0009] That is, the denatured protein solubilizing solution according to claim 1 is characterized by containing a surfactant, a reducing agent, and water.

[0010] The invention of claim 2 is characterized in that, in the denatured protein solubilizing solution according to claim 1, the surfactant component comprises any one of sodium dodecyl sulfate at a concentration of 1% to 10%, Triton X at a concentration of 1% to 10%, or Tween 80 at a concentration of 1% to 10%.

[0011] The invention of claim 3 is characterized in that in the denatured protein solubilization liquid according to claim 1 or 2, the reducing agent component is any one of 1-thioglycerol at a concentration of 0.1% or more and less than 10%, thiourea at a concentration of 2.5% or more and less than 5%, dithiothreitol at a concentration of 0.1% or more and less than 10%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.5% or more and less than 10%, and has a pH of 4 or more and 8 or less.

[0012] The invention of claim 4 is characterized in that, in the denatured protein solubilization liquid according to claim 1 or 2, the reducing agent component is any one of 1-thioglycerol at a concentration of 0.1% or more and less than 9%, thiourea at a concentration of 2.5% or more and less than 5%, dithiothreitol at a concentration of 0.1% or more and less than 10%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.1% or more and less than 9%, and has a pH of more than 8 and less than 12.

[0013] The invention of claim 5 is characterized in that, in the denatured protein solubilization liquid according to claim 1 or 2, the reducing agent component contains any one of 1-thioglycerol at a concentration of 0.1% or more and less than 2%, thiourea at a concentration of 2.5% or more and less than 5%, N,N-dimethyl-2-mercaptoethylammonium hydrochloride at a concentration of 0.1% or more and less than 6%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.1% or more and less than 6%, and has a pH of 12 to 14. Effect of the Invention

[0014] According to the present invention, heat-denatured proteins can be solubilized. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The denatured protein solubilizing solution according to the present invention is capable of solubilizing heat-denatured proteins in order to accurately evaluate the cleaning performance of equipment cleaned in an actual cleaning process.

[0016] The cleaning evaluation process consists of two steps: (1) extracting the proteins remaining on the equipment using an extraction solution, and (2) measuring the proteins present in the extracted solution. In this regard, companies may be contracted to carry out the entire cleaning evaluation process from extraction to measurement, or medical facilities may only carry out the extraction and outsource the measurement to a company.

[0017] Therefore, taking into consideration the fact that extraction will be performed in a medical facility, it is desirable that the time required for extraction be as short as possible, and it is necessary that the extraction liquid and extraction method do not require a long time or are not laborious.

[0018] Therefore, the present inventors have developed a solution that can easily extract denatured proteins without requiring any special equipment, specifically, a denatured protein solubilization solution that contains a surfactant, a reducing agent, and water.

[0019] Examples of surfactant components include Brji35, 56, 58, Emulgen911, 913, Nonidet P40 (NP40), octyl β-D-glucoside, octyl β-D-thioglucopyranoside, dodecyl β-D-maltoside, Triton X, Tween 20, 80, sodium cholate, sodium deoxycholate, sodium dodecyl sulfate (SDS), CHAPS, and CHAPSO.

[0020] Among these, it is preferable that the surfactant component contains sodium dodecyl sulfate at a concentration of 1% to 10%, Triton X at a concentration of 1% to 10%, or Tween 80 at a concentration of 1% to 10%, because this can further solubilize heat-denatured proteins.

[0021] On the other hand, examples of reducing agent components include substances having a thiol bond, a sulfide bond, or a disulfide bond, or thiourea, urea, 1-thioglycerol, ethaneaminothioglycolate, 3-mercaptopropionic acid, ammonium thioglycolate, dithiothreitol (DTT), guanidine hydrochloride, N,N-dimethyl-2-mercapto-ethylammonium hydrochloride, 2-mercaptoethanesulfonic acid sodium salt, and TCEP.

[0022] Among these, it is desirable that the reducing agent component contains any one of 1-thioglycerol at a concentration of 0.1% or more and less than 10%, thiourea at a concentration of 2.5% or more and less than 5%, dithiothreitol at a concentration of 0.1% or more and less than 10%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.5% or more and less than 10%, and that the pH is 4 or more and 8 or less. This is because heat-denatured proteins can be more solubilized.

[0023] Furthermore, it is desirable that the reducing agent component contains any one of 1-thioglycerol at a concentration of 0.1% or more and less than 9%, thiourea at a concentration of 2.5% or more and less than 5%, dithiothreitol at a concentration of 0.1% or more and less than 10%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.1% or more and less than 9%, and that the pH is higher than 8 and lower than 12. This is because the thermally denatured protein can be more solubilized.

[0024] On the other hand, it is preferable that the reducing agent component contains any one of 1-thioglycerol at a concentration of 0.1% or more and less than 2%, thiourea at a concentration of 2.5% or more and less than 5%, N,N-dimethyl-2-mercaptoethylammonium hydrochloride at a concentration of 0.1% or more and less than 6%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.1% or more and less than 6%, and that the pH is 12 to 14. This is because heat-denatured proteins can be more solubilized.

[0025] Therefore, when such a denatured protein solubilizing solution is used to extract heat-denatured protein, the heat-denatured protein can be solubilized, and the denatured protein can be easily extracted without the need for special equipment.

[0026] It has been described above that the denatured protein solubilizing solution according to the present invention can extract denatured proteins. However, the denatured protein solubilizing solution according to the present invention is not limited to this, and can solubilize heat-denatured proteins, so it also has the effect of washing denatured proteins. Therefore, the denatured protein solubilizing solution according to the present invention can also be used as a detergent for washing denatured proteins. EXAMPLES

[0027] The present invention will now be described in detail with reference to examples.

[0028] <Test Method> A test piece was prepared by applying blood to a rectangular stainless steel plate measuring 5 cm long by 1.5 cm wide, and denaturing the blood by wet heat at 95°C. The test piece was then immersed in 10 ml of each denatured protein solubilizing solution, which was adjusted to the blending concentration shown in Production Examples 1 to 876 described later using water or a pH adjuster described later, and left to stand at 40°C for 3.5 hours to extract the heat-denatured protein. The results are shown in Tables 1 to 36. The evaluation criteria were as follows: ◎: complete extraction of heat-denatured protein, ◯: 90% to 80% of heat-denatured protein was extracted, △: 70% of heat-denatured protein was extracted, and ×: heat-denatured protein was slightly or not extracted. The pH of each denatured protein solubilizing solution was adjusted using a pH adjuster (NaOH).

[0029] [Table 1]

[0030] [Table 2]

[0031] [Table 3]

[0032] [Table 4]

[0033]

Table 5

[0034]

Table 6

[0035]

Table 7

[0036]

Table 8

[0037]

Table 9

[0038]

Table 10

[0039]

Table 11

[0040]

Table 12

[0041]

Table 13

[0042]

Table 14

[0043]

Table 15

[0044]

Table 16

[0045]

Table 17

[0046]

Table 18

[0047]

Table 19

[0048]

Table 20

[0049]

Table 21

[0050]

Table 22

[0051]

Table 23

[0052]

Table 24

[0053]

Table 25

[0054]

Table 26

[0055]

Table 27

[0056]

Table 28

[0057]

Table 29

[0058]

Table 30

[0059]

Table 31

[0060]

Table 32

[0061]

Table 33

[0062]

Table 34

[0063]

Table 35

[0064] [Table 36]

[0065] <Test Results> In view of the above test results, it was found that a denatured protein solubilizing solution containing a surfactant, a reducing agent, and water can be used to solubilize heat-denatured proteins. Therefore, it was found that the use of such a denatured protein solubilizing solution can easily extract denatured proteins without requiring any special equipment.

[0066] Among these, it was found that solutions containing any of the following surfactant components were more effective at extracting denatured proteins: sodium dodecyl sulfate at a concentration of 1% to 10%, Triton X at a concentration of 1% to 10%, or Tween 80 at a concentration of 1% to 10%.

[0067] Furthermore, it was found that a solution containing any of the following reducing agent components, 1-thioglycerol at a concentration of 0.1% or more and less than 10%, thiourea at a concentration of 2.5% or more and less than 5%, dithiothreitol at a concentration of 0.1% or more and less than 10%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.5% or more and less than 10%, and having a pH of 4 or more and 8 or less, is more effective at extracting denatured proteins.

[0068] It was also found that a solution containing any of the following reducing agent components, inclusive: 1-thioglycerol at a concentration of 0.1% or more and less than 9%, thiourea at a concentration of 2.5% or more and less than 5%, dithiothreitol at a concentration of 0.1% or more and less than 10%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.1% or more and less than 9%, and having a pH of more than 8 and less than 12, is more effective at extracting denatured proteins.

[0069] On the other hand, it was found that a solution containing any of the following reducing agent components, 1-thioglycerol at a concentration of 0.1% or more and less than 2%, thiourea at a concentration of 2.5% or more and less than 5%, N,N-dimethyl-2-mercaptoethylammonium hydrochloride at a concentration of 0.1% or more and less than 6%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.1% or more and less than 6%, and having a pH of 12 or more and 14 or less, is more effective at extracting denatured proteins.

Claims

1. A denatured protein solubilization solution comprising a surfactant, a reducing agent, and water.

2. 2. The denatured protein solubilizing solution according to claim 1, comprising, as a surfactant component, any one of sodium dodecyl sulfate at a concentration of 1% to 10%, Triton X at a concentration of 1% to 10%, and Tween 80 at a concentration of 1% to 10%.

3. 3. The denatured protein solubilizing solution according to claim 1 or 2, comprising, as the reducing agent component, any one of 1-thioglycerol at a concentration of 0.1% or more and less than 10%, thiourea at a concentration of 2.5% or more and less than 5%, dithiothreitol at a concentration of 0.1% or more and less than 10%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.5% or more and less than 10%, and having a pH of 4 or more and 8 or less.

4. 3. The denatured protein solubilization solution according to claim 1 or 2, which contains, as the reducing agent component, any one of 1-thioglycerol at a concentration of 0.1% or more and less than 9%, thiourea at a concentration of 2.5% or more and less than 5%, dithiothreitol at a concentration of 0.1% or more and less than 10%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.1% or more and less than 9%, and has a pH of more than 8 and less than 12.

5. 3. The denatured protein solubilizing solution according to claim 1 or 2, comprising, as the reducing agent component, any one of 1-thioglycerol at a concentration of 0.1% or more and less than 2%, thiourea at a concentration of 2.5% or more and less than 5%, N,N-dimethyl-2-mercaptoethylammonium hydrochloride at a concentration of 0.1% or more and less than 6%, or 2-mercaptoethanesulfonic acid sodium salt at a concentration of 0.1% or more and less than 6%, and having a pH of 12 or more and 14 or less.

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