Method of estimating activity of serine protease

专利摘要:
Novel peptide derivatives which are acyl derivatives of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) or isoluminol (6-amino-2,3-dihydro-1,4-phthalazinedione) where the acyl residue is an amino acid or amino acid sequence with 2 to 4 amino acid residues, coupled with an amide bond, and where the alpha -amino group is either free or acylated, methods for their preparation and a method for laboratory diagnostics of proteases using the peptide derivatives. The derivatives are intended for quantitative determination of proteases or proteas-activity by release of luminol or isoluminol which in their free state can be brought to luminate intensely, but lose considerably in luminiscence when they are amide-bound to a peptide sequence.
公开号:SU1233806A3
申请号:SU823496751
申请日:1982-09-23
公开日:1986-05-23
发明作者:Роджер Зимонссон Лайф；Ари Элли Зало；Эрик Аурелл Лайф；Геран Клэсон Карл
申请人:Кабивитрум Аб (Фирма)；
IPC主号:

专利说明:

This invention relates to biochemistry, in particular, to a method for determining the activity of a serine protease. .
The aim of the invention is: increasing the sensitivity of the method.
The method is carried out as follows.
A substrate is added to the enzyme containing material — a labeled substance containing amimic acid derivatives of the general formula
R, (Aj), - (.- R, where R is hydrogen, aqdl;
And - valine, isoleucine, alanine,
glycine;
A - proline, phenylalanine, glycez valine, pipeccoline derivative of glutamic acid pyroglutamic acid., Leucine S alanine, glutamine ™, va acid; Glutamic acid methyl ester, arginine, isoleucine. tyrosine;
A - phenylalanine, proline, leucii series, glycine, valine. alanine;
And, - arginine, lysine; RJ - luminol, isolumicol; n - O or.
The reaction of the protease and the substrate is carried out under conditions that ensure the optimal reaction for the given; 1 winter. Depending on the degree of protease activity and substrate concentration, the reaction is carried out for 055-5 minutes
: they discontinue before the next: - by using an acid or a hen of 5 pr -1 baud 1 rt to change the pH with: c 5 to ensure the cessation of the e;
Then, the amount of the released: char1: eper is LYuM 1Nola or isoluminol, 11Pr: is divided by adding- and oxidizing agent: I-J measuring the amount of light emitted at the same time using a subcode or device. At the same time, the amount of free g arker is linearly proportional to the amount of significant, therefore the amount of light pre c. Is a measure for protease activity.
P p and; ep I, To a solution containing 100 microns, urokinase and 200 microliters of 10 And pG7u-Gily Arg-Ts2 / -HCt-, 800 microliters of gCis buffer with a pH of 9.0 was added with an ionic strength of 0.05. The mixture is incubated at 3 for 5 min. Substrate hydrolysis is stopped by addition of 20% -; acetic acid; 50 media are selected, in which the spectrofluorogactically determine the amount of released marker (isolumine), which corresponds to 0.01 gdc of urokin ase,
Example 2 To the solution according to npi jwepy, containing thrombin as the ph & rnc j, the substance with the structure is added as a substrate, n: pIEe; den. In table. I. The amount of enzyme (MKZ), which can be detected with the help of used substrates, is determined.
The results are summarized in table. one.
T,;, b l and n and I
Note: Boe - tertiary butyloxycarbonyl,
Cho - carbobenzoxy, Bz - benzoi.n, pGGi - pyroglutamic acid.
ts
PRI me R 3. Repeated with the use of serine enzymes, the method according to example 1, the substrates, according to the table. 2
Table 2
UrokinazpGhu-Ghy-Arg-IsI
- -pGhu-Ghy-Arg-pNa
Thrombin Tos-Phe-Pro-Arg-IsI
- -H-D-Phe-pip-Arg-pNa
PlasminBoc-Val-Leu-Lys-IsI
- -H-D-Val-Leu-Lys-pNa
The data presented shows that the use of the invention (examples, 3, and 5) makes it possible to increase the sensitivity of the method for determining the activity of serine proteases in comparison with the known method (examples 2, 4 and 6).
Example 4: The procedure of the method according to Example 1 is repeated. 5.0 10 M trypsin is used as an enzyme, and the following are used as substrates:
VNIIPI Order 2789/60 Circulation 490
Subscription
Random polygons pr-tie, Uzhgorod, st. Project, 4
Continued tabl,)
0.01 units 0.5 units
0.01 10 units
3.5 10 units
0.02-10 units of casein
1 1 About unit of casein
H-D-VaT-Len-Arg-IsI-2HCr; Cbo-GIy-Pro-Arg-IsI-HCI;
Boc-IIe-GIuCNCjH,) -Cly-Arg-IsI HCl;
H-Phe-Pro-Arg-IsI;
Bz-Arg-Lum-HCJ;
Bz-Arg-IsI HCI; H-D-Leu-Ser-Arg-IsI-2HC;
Cbo-Cly-Gly-Arg-Lum-AcOH;
Ac-Ala-Pro-Arg-IsI;
Suc-aja-pro-lys-isst;
H-0-Arg-VaI-Tyr-IsI-2HCl. The amount of released marker corresponds to that used in
enzyme concentration experience.
Subscription

权利要求:
Claims (1)
[1]
METHOD FOR DETERMINING SERIN PROTEASE ACTIVITY by reacting an enzyme-containing material with a labeled substance containing amino acid derivatives, characterized in that a labeled substance is of the general formula ^ - (AD - (AD - (AD-A, -R ,.
where R is hydrogen, acyl;
A _q - valine, isoleucine, alanine, glycine;
A - proline, phenylalanine, glycine, valine, the pipecoline derivative of glutamic acid, pyroglutamic acid, leucine, alanine, glutamic acid, glutamic acid methyl ester, arginine, isoleucine, tyrosine;
A - phenylalanine, proline, leucine, <© serine, glycine, valine, alanine;
A. - arginine, Lysine;
F _g - luminol, isoluminol;
η ’- 0 or 1.
>
SM i 23 3806

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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SE8005940|1980-08-25|

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