Photoelectric and Spectral Responses of Bacteriorhodopsin Modified by Carbodiimide and Amine Derivatives

Abstract

Photoelectric activity and photocycle kinetics of bacteriorhodopsin successively treated with a carbodiimide and several amine derivatives have been studied. Both spectral and electrical responses were initiated by a 15‐ns laser flash inducing a single turnover of bacteriorhodopsin. Photocycle kinetics were measured in a suspension of purple sheets. Photoelectric potential generation was monitored by a voltmeter, using a phospholipid‐impregnated collodion film covered on one side with purple sheets or proteoliposomes. It is shown that in bacteriorhodopsin treated with a carbodiimide derivative and ethylenediamine or methyl‐ o ‐arginine, significant acceleration of the millisecond phase of the photoelectric response is observed, whereas the kinetics of the microsecond phase remain unchanged. This effect is accompanied by an increase in the fast (τ 2 ms) component of the 412‐nm intermediate decay. Methylamine‐modified bacteriorhodopsin shows no such changes in electric and spectral kinetics. Treatment with ethylenediamine or arginine, rather than methylamine, desensibilizes, completely or partially, photoelectric response to inhibition by cationic effectors such as La 3+ or by increase in the H + concentration. At the same time modification sensibilizes this response to the inhibiting effect of an anion, citrate. Dark adaptation of bacteriorhodopsin modified by ethylenediamine or arginine slows down both microsecond and millisecond electrogeniac phases, whereas that of untreated or methylamine modified bacteriorhodopsin does not. Proteoliposomes of ethylenediamine‐modified bacteriorhodopsin generate a photoelectric potential of opposite direction (interior negative) as compared with those of unmodified or methylamine‐modified bacteriorhodopsin. It is concluded that the modifications by ethylenediamine and arginine change the decomposition of the 412‐nm intermediate in such a fashion that a portion of this intermediate decays faster. This results in accelerating those electrogenic phases which are associated with the 412‐nm intermediate decay. Such an effect may be due to replacement of negatively charged groups in the bacteriorhodopsin polypeptide chain by positively charged ones. Different methods for measurement of changes in the bacteriorhodopsin activity are discussed. The conclusion is made that the analysis of the kinetics of photoelectric potential generation in the purple sheet‐collodion film system is a sensitive probe for changes in bacteriorhodopsin activity.